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Studies on the low molecular weight RNA bound to e. coli ribosomes Jacobs, Morley 1972

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\\7ZA  STUDIES ON THE LOW MOLECULAR WEIGHT RNA BOUND TO  COLI RIBOSOMES  by  MORLEY JACOBS B.Sc. Hons., U n i v e r s i t y M.Sc,  University  o f Manitoba, 1964 o f Manitoba, 1967  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY  In The Department o f B i o c h e m i s t r y  We a c c e p t t h i s  t h e s i s as conforming t o the r e q u i r e d standard  The U n i v e r s i t y  of B r i t i s h  Columbia  J u l y , 1971 Revised January, 1972  In p r e s e n t i n g t h i s t h e s i s  in p a r t i a l  f u l f i l m e n t o f the r e q u i r e m e n t s  an advanced degree at the U n i v e r s i t y of B r i t i s h C o l u m b i a , I agree the L i b r a r y I further  s h a l l make i t f r e e l y  available for  agree t h a t p e r m i s s i o n f o r e x t e n s i v e  r e f e r e n c e and copying of t h i s  of  this thesis for  written  It  i s understood that copying or  thesis  \  Department  of  The U n i v e r s i t y o f B r i t i s h Columbia Vancouver 8, Canada  or  publication  f i n a n c i a l g a i n s h a l l not be a l l o w e d w i t h o u t my  permission.  that  study.  f o r s c h o l a r l y purposes may be g r a n t e d by the Head of my Department by h i s r e p r e s e n t a t i v e s .  for  i ABSTRACT Ribosomes were r e a d i l y prepared from The  coli B cells a b i l i t y o f these  varied with  ribosomes t o support  t h e method o f p r e p a r a t i o n .  too, v a r i e d with  procedures  grown t o the m i d - l o g o r l a t e l o g phase.  w e i g h t RNA (LMWRNA) bound t o these it,  by v a r i o u s  protein synthesis  The low m o l e c u l a r  ribosomes was s t u d i e d and  t h e type o f p r e p a r a t i o n .  After  these  i n i t i a l s t u d i e s , t h e procedure which e n t a i l e d seven e x t r a c t i o n s with  0.5-1.0 M NH^Cl i n t h e presence o f v a r y i n g l e v e l s o f  magnesium c o n c e n t r a t i o n was used i n a l l f u r t h e r experiments f o r t h e p r e p a r a t i o n o f ribosomes  (WRib).  Attempts were made t o remove a l l the tRNA bound t o WRib. Treatment w i t h ribosomal  HIOi,  r e s u l t e d i n a complete d i s r u p t i o n o f  s t r u c t u r e and was abandoned.  ment f a i l e d  (PM) t r e a t -  t o remove a l l t h e bound tRNA b u t r e s u l t e d i n a  more a c t i v e p r e p a r a t i o n . w i t h 0.1 mM M g  + +  Incubation  0.1 mM M g  + +  o f these PM-treated WRib  n o t o n l y removed a l l t h e bound tRNA b u t a l s o  resulted i n i n a c t i v e preparations. with  Puromycin  Treatment o f the WRib  alone gave i d e n t i c a l r e s u l t s .  s u b u n i t s were found t o be d e v o i d o f bound tRNA. s u b s t i t u t e these s u b u n i t s  Ribosomal Attempts t o  i n a p r o t e i n - s y n t h e s i z i n g system  i n p l a c e of whole ribosomes  (WRib)  failed.  The LMWRNA bound t o WRib was f r a c t i o n a t e d by a number of techniques  - Sephadex G-100 chromatography, DEAE-Sephadex  A-50, and p r e p a r a t i v e g e l e l e c t r o p h o r e s i s .  The o p t i c a l  d e n s i t y p a t t e r n s o f t h e f r a c t i o n a t i o n s showed only d i f f e r e n c e s , however, a c r y l a m i d e  small  g e l electrophoresis studies  ii  o f the peak f r a c t i o n s i n d i c a t e d t h a t chromatography on Sephadex G-100 was the method o f c h o i c e f o r the s e p a r a t i o n o f  LMW-  RNA. The tRNA bound t o WRib was f u l l y c h a r a c t e r i z e d .  coli  WRib have bound tRNA which has a c c e p t o r a c t i v i t y f o r a l l the amino a c i d s t e s t e d .  The amount o f c h a r g i n g v a r i e d from one  tRNA s p e c i e s t o another  - those  f o r tryptophan  were bound i n the l a r g e s t amounts. r e s u l t s cannot,  The t o t a l amount o f  tRNA bound to WRib amounted t o approximately  bound r e p r e s e n t e d  (rRNA).  1.3-1.7% o f the  S i m i l a r l y the amount o f 5S RNA  1.5-2.8% o f the t o t a l rRNA.  ages a r e e q u i v a l e n t t o 0.9-1.3 molecules molecule  o f 5S RNA.  These p e r c e n t -  o f tRNA bound p e r  Another s p e c i e s o f rRNA, 4.5S RNA  found bound t o the WRib b u t i n s m a l l amounts. 5S RNA p r e p a r a t i o n s c o n t a i n e d t h i s  virtually  Most o f the  from a p y r i m i d i n e -  (ATCC13135) grown i n the presence  u r a c i l , was exchanged w i t h u n l a b e l e d tRNA. 5.7 molecules  was  RNA.  The tRNA bound t o WRib, prepared r e q u i r i n g mutant  methionine  The s i g n i f i c a n c e o f these  as y e t , be e x p l a i n e d .  t o t a l r i b o s o m a l RNA  and  o f H3  A f t e r exchange  o f tRNA were bound per molecule  o f 5S RNA and  a l l o f the l a b e l e d tRNA had been removed.  These  exchanged WRib were no l o n g e r a c t i v e i n a p r o t e i n - s y n t h e s i z i n g system.  The tRNA from these WRib was f r a c t i o n a t e d on a BD-  c e l l u l o s e column. throughout  The r a d i o a c t i v i t y was evenly  spread  the s a l t g r a d i e n t b u t a peak was i s o l a t e d i n the  ethanol gradient. be accounted  The r a d i o a c t i v i t y i n t h i s peak r e g i o n may  f o r by the presence  o f 4.5S RNA.  The r e s u l t s  I l l  suggest t h a t a l l tRNAs are bound t o the WRib to the same degree and i n d i r e c t l y  supports the r e s u l t s r e p o r t e d i n the  l i t e r a t u r e o f the non-existence o f a s p e c i f i c c h a i n - t e r m i n ating  tRNA.  iv  ACKNOWLEDGEMENTS I wish to thank my research supervisor Dr. Gordon M. Tener f o r enthusiastic support, valuable suggestions and discussions during the course of t h i s work.  My associations  with the members of t h i s laboratory have been most h e l p f u l and I wish to acknowledge the debt I owe to them.  V  TABLE OF CONTENTS Abstract  PAGE i  Acknowledgements  iv  T a b l e o f Contents  v  L i s t of Tables  viii  and C h a r t s  L i s t of Figures  ix  Abbreviations  x i i  L i s t of Buffers  xiii  Dedication  xiv  Introduction  1  The Ribosome  1  Ribosomal RNA  3  Heterogeneity  o f Ribosomal RNA  7  Ribosomal P r o t e i n s  8  Stoichiometry  9  o f Ribosomal P r o t e i n s  RNA-Protein I n t e r a c t i o n and t h e I n t e r n a l O r g a n i z a t i o n o f t h e Ribosome  12  B i n d i n g o f tRNA t o Ribosomes  14  (a) N o n - s p e c i f i c B i n d i n g o f Free tRNA  14  (b) Non-enzymatic S p e c i f i c B i n d i n g o f Aminoacyl-tRNA  17  (c) E n z y m e - S p e c i f i c tRNA  22  B i n d i n g o f Aminoacyl-  (d) Summary  22  The Ribosome and P r o t e i n S y n t h e s i s  24  Release F a c t o r s and t h e Mechanism o f Termination  28  Ribosomal S t r u c t u r e and F u n c t i o n  33  vi  PAGE O u t l i n e of the Problem  43  Thesis Proposal  44  M a t e r i a l s and Methods  45  Chemicals  45  P r e p a r a t i o n of E ^ c o l i B  45  P r e p a r a t i o n o f E ^ c o l i B Mutant 12632  46  P r e p a r a t i o n of E ^ c o l i B. Ribosomes  48  Assay f o r Ribosomal A c t i v i t y and Amino Acid Incorporation  50  Assay f o r Amino A c i d A c c e p t o r  51  Activity  P r e p a r a t i o n of E ^ c o l i Aminoacyl-tRNA Synthetases  52  (a) Treatment DEAE-Cellulose  52  Column  (b) Treatment on Sephadex G-25 P r e p a r a t i o n of B e n z o y l a t e d  Column  53  DEAE-Cellulose  54  Treatment o f Ribosomes w i t h Puromycin  55  Treatment of Ribosomes w i t h P e r i o d a t e  56  A n a l y t i c a l Polyacrylamide phoresis  56  Gel  Electro-  Preparative Polyacrylamide phoresis  Gel E l e c t r o -  60  P r e p a r a t i o n o f 30S Ribosomes  Subunits  62  and  50S  from  Preparation of CsCl Gradient  63  I s o l a t i o n of T o t a l RNA  from Ribosomes  63  P r e p a r a t i o n of High and Low M o l e c u l a r Weight RNA from Ribosomes  64  Results  68  vii  PAGE The  Activity  o f Ribosome P r e p a r a t i o n s  A t t e m p t s t o Remove Bound tRNA f r o m Ribosome (a)  68 70  Puromycin  70  (b) P e r i o d a t e  72  (c) D i a l y s i s  against  S t u d i e s on t h e 30S Subunits Characterization  0.1  and  mM  50S  Magnesium Ribosomal  o f the Ribosomes  O t h e r T e c h n i q u e s Used t o F r a c t i o n a t e Low M o l e c u l a r W e i g h t RNA (a)  Preparative  Gel Electrophoresis  (b) DEAE-Sephadex C h r o m a t o g r a p h y  75 75  79 91 91 96  F r a c t i o n a t i o n o f R i b o s o m a l RNA by Preparative Gel Electrophoresis  100  Exchange Experiments  104  Discussion  119  Bibliography  146  Appendix  16 3  viii  LIST OF TABLES AND CHARTS TABLE I  PAGE Amino a c i d a c c e p t o r a c t i v i t y o f ribosomes p r e p a r e d by d i f f e r e n t methods  67  II  D i s t r i b u t i o n o f RNA i n E ^ c o l i ribosomes  82  III  S p e c i f i c amino a c i d a c c e p t o r a c t i v i t y o f tRNA bound t o E ^ c o l i ribosomes  85  IV  D i s t r i b u t i o n o f low m o l e c u l a r weight RNA i n E. c o l i ribosomes i s o l a t e d by Sephadex G-100 chromatogr aphy  88  V  Exchange o f l a b e l l e d tRNA bound t o ribosomes w i t h u n l a b e l l e d tRNA  VI  D i s t r i b u t i o n o f l a b e l l e d H-RNA i n E ^ c o l i ribosomes a f t e r exchange w i t h u n l a b e l l e d tRNA  coli  3  107 112  CHART I II  Preparation of E^ c o l i  ribosomes  Procedures f o r p r e p a r i n g  c o l i ribosomes  47 66  ix  LIST OF FIGURES FIGURE  PAGE  1  Photograph of p o l y a c r y l a m i d e g e l e l e c t r o p h o r e s i s p a t t e r n o f low m o l e c u l a r weight RNA o b t a i n e d from d i f f e r e n t p r e p a r a t i o n s of E^_ c o l i ribosomes  69  2  Photograph of p o l y a c r y l a m i d e g e l e l e c t r o p h o r e s i s p a t t e r n s of low m o l e c u l a r weight RNA o b t a i n e d from p u r o m y c i n - t r e a t e d E^_ c o l i ribosomes  71  3  Photograph of p o l y a c r y l a m i d e g e l e l e c t r o p h o r e s i s p a t t e r n s of low m o l e c u l a r weight RNA o b t a i n e d from magnesium-treated E. c o l i ribosomes  73  4  Sedimentation p a t t e r n of E ^ c o l i ribosomes on a 10-30% d i s c o n t i n u o u s sucrose g r a d i e n t  74  5  Photograph of p o l y a c r y l a m i d e g e l e l e c t r o p h o r e s i s p a t t e r n s of low m o l e c u l a r weight RNA o b t a i n e d from E ^ c o l i r i b o s o m a l s u b u n i t s  76  6  Sedimentation p a t t e r n of E ^ c o l i r i b o s o m a l subunits i n a CsCl s o l u t i o n  77  7  E l u t i o n p a t t e r n of commercial E ^ c o l i tRNA from Sephadex G-100  78  8  Photograph o f p o l y a c r y l a m i d e g e l e l e c t r o p h o r e s i s p a t t e r n s of d i f f e r e n t f r a c t i o n s of commercial E ^ c o l i tRNA chromatographed p r e v i o u s l y on a Sephadex G-100 column  80  9  E l u t i o n p a t t e r n of t o t a l r i b o s o m a l RNA E. c o l i on Sephadex G-100  81  from  10  Photograph of p o l y a c r y l a m i d e g e l e l e c t r o p h o r e s i s p a t t e r n s of peaks I I and I I I as designated i n Table I I  84  11  E l u t i o n p a t t e r n of the low m o l e c u l a r weight RNA bound to E ^ c o l i ribosomes from Sephadex G-100  86  12  Photograph of p o l y a c r y l a m i d e g e l e l e c t r o p h o r e s i s p a t t e r n s of the peak f r a c t i o n o f each peak r e g i o n of F i g u r e 11  89  X  FIGURE  PAGE  12a  Chromoscan t r a c i n g o f a m i x t u r e o f f r a c t i o n s 116 and 132 (peaks I I and I I I ) from F i g u r e 11  90  13  E l e c t r o p h o r e s i s p a t t e r n on a 10% p r e p a r a t i v e p o l y a c r y l a m i d e g e l o f 5 mg o f commercial E. c o l i tRNA  92  14  E l e c t r o p h o r e s i s p a t t e r n o f 100 mg of commerc i a l E ^ c o l i tRNA on a 10% p r e p a r a t i v e polyacrylamide g e l  93  15  Chromatography o f commercial on DEAE-Sephadex A-50  tRNA  94  16  Photograph of p o l y a c r y l a m i d e g e l e l e c t r o p h o r e s i s p a t t e r n s of the peak f r a c t i o n o f each peak r e g i o n o f F i g u r e 15  95  17  E l e c t r o p h o r e t i c p a t t e r n o f the low m o l e c u l a r weight RNA from E ^ c o l i ribosomes i n a 10% preparative polyacrylamide g e l  97  18  Photograph o f p o l y a c r y l a m i d e g e l e l e c t r o p h o r e t i c p a t t e r n s o f the peak f r a c t i o n o f each peak r e g i o n o f F i g u r e 17  98  19  Spectrum o f f r a c t i o n 26  99  20  The e f f e c t o f pH on the spectrum o f f r a c t i o n 26 (peak I) o f F i g u r e 17  101  21  Growth and pH curves o f an E ^ c o l i p y r i m i dine r e q u i r i n g mutant (ATCC 13135)  103  22  Displacement of l a b e l l e d tRNA bound t o E ^ c o l i ribosomes  105  23  Exchange of l a b e l l e d tRNA bound t o E ^ c o l i ribosomes w i t h u n l a b e l l e d tRNA  10 8  24  E l u t i o n p a t t e r n from Sephadex G-100 o f the 110 low m o l e c u l a r weight RNA bound t o l a b e l l e d E. c o l i ribosomes a f t e r exchange w i t h u n l a b e l l e d tRNA  25  Photograph o f p o l y a c r y l a m i d e g e l e l e c t r o p h o r e s i s l l l p a t t e r n s of v a r i o u s f r a c t i o n s from F i g u r e 24  coli  (peak I) o f F i g u r e 17  xi  FIGURE  PAGE  26  Chromatography on B D - c e l l u l o s e of 4S RNA bound t o E ^ c o l i ribosomes a f t e r exchange w i t h u n l a b e l l e d tRNA  114  27  Chromatography on B D - c e l l u l o s e o f 4S RNA n o r m a l l y bound t o E_^ c o l i ribosomes  115  28  Chromatography on B D - c e l l u l o s e o f commercial E. c o l i tRNA  117  xii  ABBREVIATIONS All  a b b r e v i a t i o n s and t e r m i n o l o g y used i n t h i s  are i n accordance  thesis  w i t h those n o r m a l l y accepted by t h e  J . B i o l . Chem., 246, 4 (1971) and o n l y those a b b r e v i a t i o n s not l i s t e d i n t h i s j o u r n a l a r e shown below. fMet  formylmethionine  aa-tRNA  Aminoacyl-tRNA  tRNA  Met m or f  A s p e c i f i c member o f the group o f tRNAs which accept methionine.  DF  Dissociation Factor  R factor  Release F a c t o r  TEMED  N,N,N',N'-Tetramethylethylenediamine  WRib  Washed Ribosomes  PM  Puromycin  ATA  Aurintricarboxylic Acid  PPO  2,5-diphenyloxazole  dimethyl-POPOP  1,4-bis-2-(4-methyl-5-phenyloxazolyl)benzene  DMAPN  3 -dimethylaminopropionitrile  TCA  Trichloroacetic Acid  A2  Absorbance a t 260 nm  1  6 0  A26O u n i t  One A2 6 0 u n i t i s t h a t amount o f m a t e r i a l which when d i s s o l v e d i n one ml o f s o l v e n t w i l l g i v e an absorbance o f one i n a c e l l w i t h a l i g h t path o f 1 cm.  Bis  N,N'-Methylenebisacrylamide  Mg(OAc)  magnesium a c e t a t e  BD-cellulose  Benzoylated  2  DEAE-cellulose  xiii  LIST OF BUFFERS Buffer  Components  A  lOmM T r i s , lOmM Mg(OAc) , lOmM NHifCl, lOmM mercaptoe t h a n o l , pH 7.8  B  lOmM T r i s , O.lmM Mg(OAc) ,  C  lOmM T r i s ,  D  lOmM T r i s , 5mM Mg(OAc) , 0.5-1.0M NH^Cl, pH 7.4  E  10 mM T r i s ,  F  lOmM T r i s , lOmM Mg(OAc) , lOmM NH^Cl, pH 7.6  G  10 mM T r i s , 0.2M KC1, pH 7.4  H  20mM T r i s , lOmM c y s t e i n e , pH 7.4  I  lOmM T r i s , O.lmM Mg(OAc) , pH 7.6  K  2  0.5-1.0M NHijCl, pH 7.4  2  ImM Mg(OAc) , 0.5-1.0M NHi»Cl, pH 7.4 2  2  lOmM Mg(OAc) , 0.5-1.0M NHi»Cl, pH 7.4 2  2  2  5mM T r i s , O.lmM Mg(OAc)  2 l  pH 7.3  lOmM Mg(OAc) , 0.5% sodium d o d e c y l  L  lOmM T r i s , PH 7.6  M  lOmM T r i s , 5mM Mg(OAc) , 250mM NH4CI, pH 7.8  N  lOmM T r i s ,  2  2  lOmM Mg(OAc) , 1.0M NH^Cl, pH 7.8 2  sulfate,  xiv DEDICATION To my p a r e n t s who  i n s t i l l e d me w i t h the d r i v e and  i n c e n t i v e , to my d e a r e s t Bev who  gave me  c o n f i d e n c e and l o v e and t o Stephanie  encouragement,  for priority  lessons  1 INTRODUCTION The  amino acid sequence o f a p a r t i c u l a r protein i s  s p e c i f i e d by the sequence of nucleotides i n a p a r t i c u l a r segment of the deoxyribonucleic acid step the DNA i s transcribed  (DNA).  In the t r a n s c r i p t i o n  into a ribonucleic acid (RNA)  intermediate c a l l e d messenger RNA (mRNA), which has a r i b o nucleotide sequence complementary to that of the deoxyribonuc l e o t i d e sequence o f one o f the strands of the DNA serving as template (64).  The t r a n s l a t i o n steps follows.  becomes attached to cytoplasmic ribonucleoprotein  The mRNA particles  (ribosomes) which are the s i t e s o f protein synthesis, and there i t determines the order of linkage of amino acids a s p e c i f i c protein  (65-67) .  into  During t r a n s l a t i o n a group of  three adjacent nucleotides i n the mRNA (codon) s p e c i f i e s which amino acid i s to be linked to the growing peptide chain. There are 64 possible codon t r i p l e t s and 61 of these specify a p a r t i c u l a r amino acid (68).  The other three codons, UAA,  UAG and UGA are c a l l e d nonsense codons since they a c t as chain terminators which signal the end of a genetic message. Each amino acid i s joined s p e c i f i c a l l y to one of the codonrecognizing molecules known as transfer RNA (tRNA).  Each  tRNA molecule has i t s own t r i p l e t of bases, c a l l e d an a n t i codon, that recognizes the relevant  codon on the mRNA by  p a i r i n g bases with i t . The  Ribosome Zamecnik and his co-workers established  the central role  2  of ribosomes i n protein synthesis (although e a r l i e r c y t o l o g i c a l studies had shown ribosomes to be the s i t e of protein synthesis), and i n addition, discovered most of the components involved i n i n v i t r o protein synthesizing systems, such as tRNAs and aminoacyl-tRNA synthetases  (1).  However, mRNA  had not been discovered and i t was thought that ribosomal RNAs (rRNAs) were the templates on the ribosomes.  f o r the proteins synthesized  Thus i t was hoped that studies on the  structure of ribosomes and rRNA would give some clue as to the mechanism of information transfer from genes to proteins. About 1957 the f i r s t systematic studies on the i s o l a t i o n and c h a r a c t e r i z a t i o n of ribosomes were i n i t i a t e d (2-5) . studies were done on ribosomes from  These  c o l i and established  the following basic information : (a) Ribosomes isolated and p u r i f i e d i n the presence of 10 mM M g c o e f f i c i e n t of 70S.  ++  have a sedimentation  A f t e r treatment with puromycin which  removes nascent peptides, these ribosomes d i s s o c i a t e into 3OS  and 50S subunits upon lowering M g  ++  concentration (1 mM  or less) (2,6); however, i f nascent peptide i s present, complete removal of M g  ++  with EDTA i s required (69).  (b) The  50S and 30S ribosomal subunits have a p a r t i c l e weight of 1.8 x 10 and 0.85 x 10 daltons respectively (6). 6  6  (c) Both 50S  and 30S subunits contain about 2/3 RNA and 1/3 p r o t e i n . (d) The 50S subunit contains one molecule of RNA, 23S RNA (The presence of one molecule of 5S RNA was discovered l a t e r (55)) , and the 30S subunit contains one molecule of RNA, 16S RNA (7).  3  Subsequent progress i n the study of ribosomes, however, lagged f a r behind progress i n other areas of molecular biology. This was p a r t l y due to the discovery of mRNA which diverted attention from ribosomes, and p a r t l y due to the d i f f i c u l t i e s caused by the s t r u c t u r a l complexity of ribosomes.  For example,  30S subunits contain about 20 d i f f e r e n t proteins and subunits contain about 30 to 35 d i f f e r e n t proteins.  50S However,  i t was soon evident that the detailed mechanism of protein synthesis could not be elucidated without knowledge of the structure and function of ribosomes. role f o r ribosomes was suggested  Moreover, an active  i n the codon-anticodon recognition process  (8).  Thus serious i n t e r e s t i n the ribosome was  revived and the l a s t several years have witnessed rapid progress i n the study of them. Ribosomal  RNA  Current evidence suggests that the 30S ribosomal subunit contains one molecule of 16S RNA with a molecular weight of 5.5 x 10  s  while the 50S ribosomal subunit contains one mole-  cule of 23S RNA  with a molecular weight of 1.1 x 10  6  molecule of 5S RNA with a molecular weight of 4 x 10" 44).  Since the molecular weight of 23S RNA  that of the 16S RNA, the 23S RNA  and one (7, 42,  i s about twice  there have been frequent claims that  i s a dimer of a "16S" RNA molecule which i s  i d e n t i c a l or very s i m i l a r to the 16S RNA molecule found i n 30S ribosomal subunits.  Several observations o r i g i n a l l y  supported t h i s idea (7, 45-47).  4  One can now exclude t h i s claim.  The 23S and 16S rRNAs  have been shown to be d i f f e r e n t i n base composition (44), base sequence as judged by DNA-RNA h y b r i d i z a t i o n (48) , o l i g o nucleotide patterns obtained a f t e r enzymatic digestion (49), methylated oligonucleotides obtained after T i RNase digestion (47) and i n t h e i r 5'-terminal sequences (50).  I t was also  found that the "16S RNA prepared from 23S RNA according to n  the method of Midgley  (45) could not replace 16S rRNA i n the  r e c o n s t i t u t i o n of 30S ribosomal subunits.  Thus, i t was  u n l i k e l y that the 23S rRNA was formed by simple dimerization of two 16S rRNA molecules. The claim that the SOS ribosomal subunit contains two RNA chains i s also d i f f i c u l t to accept.  F i r s t , the conversion  of 23S RNA into smaller RNAs which had been claimed by several workers could not be observed under conditions minimizing nuclease contamination  (51).  Second, c a r e f u l studies done by  Stanley and Bock (127) revealed no non-covalent bond i n the 23S rRNA molecule.  F i n a l l y Leppla (52) measured the number  of chain terminal bases i n the 23S rRNA molecule and obtained r e s u l t s which were consistent with one chain terminus per 23S rRNA.  I t therefore appears that the 23S rRNA i s a single  polynucleotide chain with a molecular weight of 1.1 x 1 0 . 6  Although the 23S rRNA i s a single polynucleotide chain, the work of F e l l n e r and Sanger (47) strongly indicated that the molecule was made up of two sections which may be i d e n t i c a l or very s i m i l a r i n their base sequence.  One of t h e i r  5  suggestions was that the 23S rRNA c i s t r o n had arisen by a "gene duplication" mechanism during evolution.  Whether the  possible existence of two i d e n t i c a l or s i m i l a r parts i s related to the functions  of 23S rRNA (53) i s not c l e a r .  A related  subject i s the problem of sequence homology between 16S and 23S rRNAs.  Despite clear-cut evidence f o r a sequence d i f f e r -  ence between E_^_ c o l i 16S and 23S rRNAs, DNA-RNA h y b r i d i z a t i o n experiments have shown that 16S and 23S rRNAs compete f o r the same DNA s i t e s to a great extent (54) .  This suggests that  DNA c i s t r o n s f o r both 16S and 23S RNA have evolved by gene d u p l i c a t i o n s t a r t i n g from a common gene.  Alternatively,  p a r t i a l sequence homology may r e f l e c t a common (unknown) function performed by parts of both 16S and 23S RNAs.  On the  other hand, DNA-RNA h y b r i d i z a t i o n experiments done with B a c i l l u s megaterium and B a c i l l u s s u b t i l i s showed a complete lack of sequence homology (48) .  I t i s not clear i f the  observed discrepancy was due to a difference i n techniques used or to the difference i n b a c t e r i a l  species.  Maturation of rRNAs i n prokaryotes and eukaryotes has a common feature;  the process seems to involve the cleavage of  precursor molecules larger than the mature rRNAs but there are differences.  In eukaryotes the two rRNAs are produced by  s p l i t t i n g a single large precursor molecule whereas i n b a c t e r i a the two rRNAs are derived  from two discrete precursors each  only s l i g h t l y larger than the mature species (206) .  6  The 50S ribosomal subunit contains one molecule of 5S RNA  i n addition to 23S rRNA (55).  The 5S RNA  amino acids and i s thus d i f f e r e n t from tRNA.  does not accept I t i s not a  random breakdown product of 16S or 23S rRNA but appears to be a genuine ribosomal component present i n a l l 50S subunits of various o r i g i n s .  To date l i t t l e , i f any i s known of the  functional role of 5S RNA  although K i r t i k a r and K a j i  have shown that the addition of 5S RNA  stimulated incorpora-  t i o n of amino acids into protein directed by RNA MS-2.  (128)  from phage  Siddiqui and Hosokawa (131) have suggested that 5S  rRNA may have a role i n the s p e c i f i c binding of tRNA to r i b o somes.  Raacke (132) has proposed a c l o v e r l e a f conformation  for 5S RNAs.  The model proposes three functions f o r 5S RNA  :  (1) the binding of 5S RNAs to 50S ribosomes by means of a unique and universal base sequence,  (2) the joining of 30S  to 50S ribosomes i n a s p e c i e s - s p e c i f i c manner, (3) the binding of  tRNA through s p e c i f i c base p a i r i n g and Mg  bridges.  Recently Jordan (248) has found that the most exposed part of the  c o l i 5S RNA  (as determined by T i RNase cleavage) has a  sequence complementary to the -GTUC sequences found i n a l l G  tRNAs thus f a r sequenced.  Although t h i s would appear to  strengthen the case f o r a function of 5S RNA  involving i n t e r -  action with tRNA i n the ribosome, this sequence i n tRNA i s i n the  most protected loop.  Therefore i n order f o r i n t e r a c t i o n  to occur the tRNA would have to undergo a conformatioal change which would involve ring opening. also been proposed  (42, 133-135) .  Other models have  The 5S RNA  from E_j_ c o l i  7  consists of 120 nucleotides and lacks methylated or unusual bases i n contrast to other rRNAs or tRNAs (42) . tide sequence of 5S RNA from  coli  The nucleo-  (42) and from mammalian  KB c e l l s (129) has been established, and i t i s i n t e r e s t i n g to note that a part of the nucleotide sequence i s duplicated i n each of the two 5S RNA species.  Preliminary studies also  indicate that the sequence of 5S rRNA from two mouse c e l l l i n e s i s the same as that found i n human KB c e l l s Recently  (136) .  the nucleotide sequence of Pseudomonas fluorescens  5S RNA has been elucidated and found to contain many s i m i l a r i t i e s to the  c o l i 5S RNA (209).  No base sequence homology  has been found between 5S RNA and 16S or 23S rRNA using the technique of DNA-RNA h y b r i d i z a t i o n (56) .  Recently Monier's  group (130) demonstrated the presence i n exponentially growing E. c o l i c e l l s of a precursor of 5S RNA. longer by 1-3 nucleotides.  A s i m i l a r precursor was synthe-  sized i n the presence of chloramphenicol Heterogeneity  The precursor was  (247).  of Ribosomal RNA  DNA-RNA h y b r i d i z a t i o n experiments have c l e a r l y shown that genes f o r 16S and 23S rRNA are present i n multiple copies  (137-140).  For instance, Spadari and Ritossa (137)  confirmed the existence of 6 genes f o r 23S rRNA and 6 f o r 16S rRNA f o r each Ej_ c o l i chromosome.  Because of t h i s redun-  dancy, i t i s quite possible that the genes f o r 16S rRNA or those f o r 23S rRNA are not homogeneous and that there are several chemically d i f f e r e n t species of 16S rRNA and 23S rRNA  8  (46, 50, 57).  At l e a s t three d i f f e r e n t l o c i for 5S  c i s t r o n s have been l o c a l i z e d i n E_j_ c o l i  (141) .  RNA  The question  of the possible heterogeneity of rRNAs i s important, since i t implies the heterogeneity of each of the ribosomal  subunits  and i s possibly related to some functional d i f f e r e n t i a t i o n among ribosomes. On the other hand, the sequence analysis of  methylated  oligonucleotides of rRNA done by F e l l n e r and Sanger  (47)  showed that many long oligonucleotides with unique base sequences and chain lengths up to eleven occurred i n one, or four moles per mole of RNA, than one mole.  two  and never i n an amount less  Thus, both 16S and 23S rRNA from  c o l i are  l a r g e l y homogenous, at l e a s t with respect to the base sequences around methylated  nucleotides.  Further extensive analysis of  fragments of 16S rRNA performed by F e l l n e r et al^. strongly indicated homogeneity of 16S rRNA (58, 142).  Completion of  such base sequence analysis w i l l undoubtedly give a more convincing answer to the question of rRNA heterogeneity. should be noted that the base sequence of 5S RNA has proved the homogeneity of t h i s RNA  from  It coli  species (42) , despite  the m u l t i p l i c i t y of c i s t r o n s for t h i s RNA  i n the b a c t e r i a l  genome (48) . Ribosomal Proteins I t appears that the 30S ribosomal subunit of contains about 21 proteins. Kurland's  (33, 210)  coli  This has been confirmed by both  and Nomura's groups (38).  The average  9  molecular weight i s 12,400 per protein. Traut and his co-workers (34) i s o l a t e d 36 proteins from the 50S ribosomal subunits of  c o l i and concluded that the  number of 50S proteins could be between 34 and 38. Kurland and h i s co-workers concluded that the number of 50S proteins i s between 25 and 31 with an average molecular weight of 15,200.  Kaltschmidt  and Wittman found as many as 34 ribosomal  proteins on the 50S subunit  (123).  I t i s s i g n i f i c a n t to note  that there i s no protein common to both 50S and 30S p a r t i c l e s . This conclusion was obtained by both chemical and immunologic a l studies (33-36, 121-122, 124-126).  The r e s u l t s  the conclusion that there i s no extensive  favour  s t r u c t u r a l homology  among ribosomal proteins ( r P r o t e i n s ) . Stoichiometry  of Ribosomal Proteins  I t has usually been assumed that the ribosome has a defined structure and that the ribosome population i s homogeneous.  The heterogeneity  of rRNA and hence of ribosome  populations was suggested by several i n v e s t i g a t o r s , but i t i s only now that Kurland's group has performed c a r e f u l studies on the stoichiometry of ribosomal proteins that the question has been brought to serious consideration  (210).  Thus, when the f i r s t experimental studies on t h i s problem by Moore's group (35) showed that most (thirteen) of the ribosomal proteins existed i n amounts corresponding to one copy per 30S p a r t i c l e , the conclusion was r e a d i l y accepted that the ribosomes were homogeneous with respect to t h e i r  10 p r o t e i n composition.  However, l a t e r work by K u r l a n d e t a l .  (36) d i d not agree w i t h t h i s c o n c l u s i o n . r i b o s o m a l p r o t e i n s and found  They examined 21  t h a t 12 o f them were p r e s e n t i n  amounts c o r r e s p o n d i n g t o about one copy per 30S p a r t i c l e , b u t t h a t 8 o t h e r p r o t e i n s were p r e s e n t i n amounts l e s s than c o p i e s per ribosome.  There was no evidence which  0.7  suggested  t h a t any 30S p r o t e i n was p r e s e n t i n amounts c o r r e s p o n d i n g t o more than 1 copy per ribosome.  They concluded  t h a t t h e r e are  two k i n d s o f p r o t e i n s ; one which they c a l l e d " u n i t i s p r e s e n t i n a l l the i s o l a t e d  protein"  30S r i b o s o m a l p a r t i c l e s and  the o t h e r which they c a l l e d " f r a c t i o n a l p r o t e i n " i s p r e s e n t i n some but not a l l the i s o l a t e d 30S p a r t i c l e s . between Kurland's  Discrepancies  and Moore's l a b o r a t o r i e s were mostly  i n the  m o l e c u l a r weight v a l u e s a s s i g n e d t o some o f the p r o t e i n s . Although work done by another group a l s o f a v o r e d the c o n c l u s i o n t h a t a l l the r i b o s o m a l p r o t e i n s e x i s t e d i n s t o i c h i o m e t r i c amounts (37) , i t now appears  t h a t the c o n c l u s i o n o b t a i n e d by  Kurland's group i s c o r r e c t , a t l e a s t w i t h r e s p e c t t o ribosomes o b t a i n e d as i n v i t r o p r e p a r a t i o n s . m o l e c u l a r weights  Recent i n v e s t i g a t i o n s o f  o f 30S r i b o s o m a l p r o t e i n s by Moore's group  has now y i e l d e d data c o n s i s t e n t w i t h t h a t o b t a i n e d by K u r l a n d and h i s c o l l a b o r a t o r s  (34). There are s e v e r a l o t h e r  facts  s u p p o r t i n g the c o n c l u s i o n o f a heterogeneous p o p u l a t i o n o f ribosomes.  First,  there i s a s t r i k i n g c o r r e l a t i o n between  p r o t e i n s c l a s s i f i e d as u n i t p r o t e i n s by K u r l a n d e t al_. and p r o t e i n s found by Nomura's group to be r e q u i r e d f o r the  11 "physical assembly" of ribosomes.  Such c o r r e l a t i o n i s  consistent with the mechanism of the ordered assembly of 30S particles  (38, 211) .  Second, Kurland and his co-workers were  able to show as much as 60% stimulation of a c t i v i t y of 30S p a r t i c l e s by incubating them with externally added 30S ribosomal proteins under the conditions optimum for r e c o n s t i tution.  Concomitant with t h i s stimulation, they observed  that some externally added proteins were incorporated  into  the p a r t i c l e s and some proteins i n i t i a l l y present i n the 30S p a r t i c l e s were released into the medium (36).  Although  i n t e r p r e t a t i o n of the observed protein exchanges must await exact i d e n t i f i c a t i o n of these exchanged proteins, the observed facts are consistent with the conclusion that the i s o l a t e d 30S p a r t i c l e s are not f u l l y active and that part of the reason f o r the i n a c t i v i t y i s a deficiency i n some ribosomal proteins i n some of the 30S p a r t i c l e s .  An a l t e r n a t i v e explanation  i s the  steady-state hypothesis of Kurland (210) who postulates a functional cycle i n which the complement of f r a c t i o n a l proteins on a ribosome changes as a given ribosome proceeds through the d i f f e r e n t operational modes. One example of such a cycle would e n t a i l d i f f e r e n t sets of f r a c t i o n a l proteins  associated  with the ribosomes during chain i n i t i a t i o n , propagation, termination and a r e s t mode.  Some of the proteins which are  involved i n such a cycle might be required for protein synthesis by a l l ribosomes. In contrast to the 30S ribosomal proteins, most of the  12 50S ribosomal proteins appear to be present i n amounts corresponding to one copy per 50S p a r t i c l e .  Traut et al^. (34)  found that 31 of the 50S ribosomal proteins existed i n stoichiometric amounts and only 2, or possibly 4, of the 50S ribosomal proteins existed i n amounts that were much less than one copy per p a r t i c l e .  Kurland's group has also f a i l e d to  detect any s i g n i f i c a n t heterogeneity populations  so f a r .  of 50S ribosomal  subunit  A major conclusion which has emerged from  studies on ribosomal proteins i s that, because none of the proteins e x i s t s as more than one copy per p a r t i c l e , ribosomal subunits have no symmetry.  This means that any model of  ribosome function involving s t r u c t u r a l symmetry, f o r example, the presence of two or more i d e n t i c a l s i t e s on a ribosome, can be discarded. RNA-Protein Interaction and the Internal Organization of the Ribosome I t i s possible to assemble 30S ribosomal subunits from free 16S rRNA and a mixture of about 20 d i f f e r e n t ribosomal protein molecules (38,60).  I t was found that the assembly  reaction required the presence of a s p e c i f i c RNA.  In the  absence of rRNA, no p a r t i c l e s resembling 30S ribosomal were formed.  Furthermore, neither 17S cytoplasmic  yeast nor "16S" RNA prepared from replace the 16S  subunits  rRNA from  c o l i 23S rRNA could  c o l i rRNA i n the r e c o n s t i t u t i o n .  With  these two RNAs no p a r t i c l e sedimenting at 30S was formed.  Rat  l i v e r 18S rRNA also could not replace 16S E. c o l i rRNA i n the  13 reconstitution.  These experiments c l e a r l y show that the rRNA-  ribosomal protein i n t e r a c t i o n i s s p e c i f i c and i s important f o r the o v e r a l l organization of ribosomal p a r t i c l e s .  Recently  Nomura's group has been able to reconstitute 50S ribosomal subunits of B a c i l l u s stearothermophilus (144) .  They were able  to show a p a r t i a l requirement f o r 5S RNA i n the r e c o n s t i t u t i o n of functional 50S p a r t i c l e s .  Their r e s u l t s also indicated  s p e c i f i c i t y of binding between the 23S rRNA and ribosomal proteins. Nomura's group has also shown that a l l mutational a l t e r a tions discovered  so far which i n h i b i t 30S ribosomal subunit  assembly also i n h i b i t 50S ribosomal subunit assembly, whereas many mutations which abolish 50S assembly do not appear to a f f e c t 30S assembly (145-146).  They proposed that assembly of  50S ribosomal subunits somehow depended on simultaneous assembly of 30S ribosomal subunits i n vivo, while the assembly of 30S p a r t i c l e s was independent of 50S assembly. Experiments to date strongly suggest that  single-stranded  regions are important i n the RNA-protein i n t e r a c t i o n , but do not exclude the possible a d d i t i o n a l involvement of h e l i c a l regions i n the i n t e r a c t i o n (234). The  chemical basis of the s p e c i f i c i t y demonstrated i n  the RNA-protein i n t e r a c t i o n i s s t i l l a matter of speculation. From the foregoing  discussion, i t i s clear that one cannot  make a d e t a i l e d model of ribosome structure.  The topological  r e l a t i o n s h i p of the many d i f f e r e n t molecular components i s  14 unknown.  Even the very elementary questions  teins are on the surface  as to which pro-  and which proteins are buried i n s i d e ,  or which part of the rRNA molecule i s exposed on the surface of the ribosome, are not answered.  However, i t i s believed  that a t l e a s t a part of the rRNA i s exposed and thus the ribosomal strucutre i s d r a s t i c a l l y d i f f e r e n t from the common s p h e r i c a l v i r u s structure i n which the RNA i s completely protected by an outer protein s h e l l . suggest that a conformational  Miskin e t a l .  (234)  change i n a protein, a l o c a l  rearrangement of a h e l i c a l portion of RNA, or an a l t e r a t i o n i n some protein-RNA association are some of the p o s s i b i l i t i e s that can account f o r the t r a n s i t i o n between the active and i n a c t i v e states of the ribosome (62, 63). s t r u c t u r a l models must be considered  A l l detailed  highly speculative a t  t h i s time. Binding of tRNA to Ribosomes (a) Non-Specific  Binding of Free tRNA  Both tRNA and aa-tRNA were shown i n early studies to attach to ribosomes (148, 251-252).  The tRNA-ribosome  i s stable only i n solutions of high M g  ++  complex  ion concentration.  I t i s s t i l l unknown what forces are involved i n holding the two components  together  i n a stable complex.  Presumably,  magnesium bridges are formed not only between subunits but also between the tRNA-ribosome complex.  The M g  ++  ions tend  to overcome the mutual repulsion of the phosphate groups of RNA to allow binding.  Bound tRNA w i l l not wash o f f i n high  15 Mg  + +  i o n c o n c e n t r a t i o n b u t can e a s i l y be d i s p l a c e d by f r e e  tRNA from the surrounding a f f e c t e d by c h a r g i n g  medium (148) .  tRNA w i t h  T h i s exchange i s n o t  amino a c i d s  (249-250) , by  temperature, o r by puromycin o r chloromphenicol  (148) .  The  n o n s p e c i f i c a s s o c i a t i o n o f tRNA and ribosomes takes p l a c e i n the c o l d and does n o t r e q u i r e the supernatant ATP  o r an energy source  r e a c t i n g components.  enzymes, GTP,  o t h e r than the thermal energy o f the  I f the t e r m i n a l pCpCpA sequence o f the  tRNA i s damaged t h i s b i n d i n g does n o t o c c u r .  I f the t e r m i n a l  adenosine i s removed by o x i d i z i n g d e a c y l a t e d  tRNA w i t h p e r i o -  date f o l l o w e d by treatment w i t h eyelohexylamine  (253) , the  a b i l i t y o f the tRNA t o b i n d t o the ribosomes i s d e s t r o y e d (148) .  I f the t e r m i n a l sequence i s reformed  the tRNA w i t h CTP, The  the supernatant  the a b i l i t y  (254) by i n c u b a t i n g  f r a c t i o n and ATP, o r ATP and  t o b i n d i s p a r t i a l l y r e - e s t a b l i s h e d (252) .  requirement f o r the i n t a c t pCpCpA terminus common t o a l l  tRNA m o l e c u l e s ,  i n d i c a t e s t h a t the t e r m i n a l sequence p l a y s a  d e c i s i v e r o l e i n the b i n d i n g o f tRNA t o ribosomes. binding i s s p e c i f i c  f o r 50S r i b o s o m a l  do n o t have any a f f i n i t y  subunits;  The  30S s u b u n i t s  f o r tRNA i n the absence o f mRNA.  Q u a n t i t a t i v e s t u d i e s showed t h a t there i s o n l y one b i n d i n g s i t e p e r 70S o r 50S ribosome  (148).  According  to Cannon  e t a l . (148) t h i s does n o t e l i m i n a t e the p o s s i b i l i t y o f s e v e r a l s i t e s , b u t i f t h e r e a r e , e i t h e r they a r e n o t a l l e q u i v a l e n t , o r i f they a r e e q u i v a l e n t they have the p r o p e r t y t h a t b i n d i n g tRNA t o any one weakens the b i n d i n g a b i l i t y o f  16  the o t h e r s .  They suggest two  exchange of tRNA.  models f o r the b i n d i n g  In the f i r s t  which the tRNA binds  the ribosome has  i n rapid e q u i l i b r i u m with  one  and site  on  the tRNA i n  solution.  In the second model the tRNA i s t i g h t l y bound to  a s i t e and  the complex d i s s o c i a t e s very s l o w l y .  would take p l a c e by and  f o r the b i n d i n g  t i g h t l y but loosely.  exchange  a second tRNA molecule a l t e r i n g the  d i s p l a c i n g the f i r s t .  sites  The  One  can c o n s i d e r two  : e i t h e r s i t e alone  site  equivalent  can b i n d the tRNA  i f both are f u l l , both tRNA molecules are bound  Such a s t r u c t u r e would show r a p i d exchange  associated with  the loose b i n d i n g f o r the two  only slow l o s s and  t i g h t binding with  d i s t i n c t i o n between the two the i n t e r m e d i a t e intermediate  one  models l i e s  molecules  tRNA.  The  but  basic  i n the composition  s t a t e d u r i n g the exchange process  : the  s t a t e i n the second model i s a ribosome and  bound tRNA molecules,  of  two  i n the f i r s t model i t i s a f r e e r i b o -  some. In the absence o f p r o t e i n s y n t h e s i s or mRNA, and 0.1  mM  Mg  + +  ion concentration,  i n t o t h e i r subunits tRNA i s completely in  the ribosomes are d i s s o c i a t e d  and i n the ensuing removed (148).  a c e l l - f r e e e x t r a c t of  at  coli,  process  a l l the bound  After protein synthesis a small f r a c t i o n of  the  tRNA t h a t i s bound t o the ribosomes i n high magnesium (10 becomes r e s i s t a n t t o b e i n g washed o f f the ribosomes i n magnesium  (0.1 mM).  low  T h i s amounts t o about h a l f a molecule  of tRNA per ribosome (148) .  T h i s has been i n t e r p r e t e d as  mM)  17 due to the presence of a nascent polypeptide chain on the tRNA which s t a b i l i z e s the binding of the tRNA to the ribosome. Takanami (252, 255) has also observed, using r a t l i v e r ribosomes, that the ribosomes w i l l bind tRNA on a roughly one-for-one b a s i s .  This binding i s magnesium dependent, but  after i n v i t r o protein synthesis, some of the bound tRNA remains t i g h t l y bound to ribosomes i n low magnesium.  He  has shown that t h i s t i g h t l y bound tRNA i s not covalently linked to the rRNA but rather i s attached to the end of the nascent polypeptide chain. (b) Non-Enzymatic S p e c i f i c Binding of Aminoacyl-tRNA In the presence of template RNA, tRNA attaches to r i b o somes with s p e c i f i c i t y (149) and i n h i b i t s the binding of aa-tRNA to ribosomes, presumably by competing with aa-tRNA for ribosomal binding s i t e s  (255). Phe  binding of both deacylated tRNA (250).  At high M g  TT  Poly U stimulates the and Phe-tRNA to ribosomes  i o n concentration, tRNA  ribosomes with approximately same extent as Phe-tRNA.  binds to  the same a f f i n i t y , and to the  Since both tRNA and aa-tRNA  recognize codons, the r a t i o of tRNA to aa-tRNA may sometimes regulate the rate of p r o t e i n synthesis.  I t has been shown,  however, that with addition of a soluble enzyme f r a c t i o n and GTP, the i n h i b i t i o n of Phe-tRNA binding to ribosomes by tRNA i s greatly reduced (250, 256) .  The presence of ribosomal  i n i t i a t i o n factors (257) and GTP reduces considerably the i n h i b i t i o n , due to tRNA, of AUG-dependent binding of N-fMet-  18 tRNA.  S p e c i f i c binding could be shown to occur i n s o l u t i o n  of high M g  ++  i o n concentration; a binding which was enhanced  by K* or N H i , * ions (149-152) .  This f i n d i n g does not demand  a s p e c i a l energy source other than the thermal energy of the reacting components.  The i n a b i l i t y of the mRNA-ribosome  complex to d i s t i n g u i s h between free tRNA and i t s amino acid charged form (149, 153) confirms the adaptor hypothesis (154) and demonstrates that the amino acid per se i s not involved i n the t r a n s l a t i o n step (153-155) .  With respect to the  binding forces that mediate the mRNA-dependent binding of tRNA to ribosomes, t h i s r e s u l t , i n a d d i t i o n , suggests that the 3'-hydroxyl group of the pCpCpA-terminus has no influence on the s t a b i l i t y of the complex  (153).  A f i r s t h i n t as to the number of s p e c i f i c tRNA-binding s i t e s on the ribosome came from the discovery that poly Udirected binding of phenylalanine  tRNA to 30S ribosomal  subunits i s stimulated approximately 50S ribosomal subunits at high M g  ++  twofold by the present of i o n concentration and  i n the absence of protein synthesis (156-158). i s consistent with the hypothesis  This finding  that one molecule of  aa-tRNA can bind to the 30S p a r t i c l e  (acceptor site) and that  the second binding s i t e i s generated by formation of the 70S ribosome (donor s i t e ) . Aside from the anticodon region, the remaining  part of  the tRNA-molecule may i n t e r a c t n o n s p e c i f i c a l l y with the r i b o some.  I t i s tempting to assume that the 30S subunit of the  70S ribosome has t h i s function.  In f a c t , the a b i l i t y of  19 i s o l a t e d 30S p a r t i c l e s t o b i n d i n d i c a t e s , besides  aa-tRNA i n response t o mRNA  the s p e c i f i c codon-anticodon r e l a t i o n s h i p ,  a s t r o n g n o n s p e c i f i c i n t e r a c t i o n between aa-tRNA and the 30S ribosomal subunit  (156, 159).  the s p e c i f i c aa-tRNA b i n d i n g  Furthermore, the response o f  r e a c t i o n to a large array of  i n h i b i t o r s o f p r o t e i n s y n t h e s i s , some o f which a c t s p e c i f i c a l l y on  the 30S r i b o s o m a l s u b u n i t ,  provides  some evidence f o r the  n o n s p e c i f i c a s s o c i a t i o n between the ribosome and a p a r t o f the aa-tRNA molecule which comprises base sequences o f the a n t i c o d o n Yet,  outside  region.  i t i s very  l i k e l y t h a t both r i b o s o m a l s u b u n i t s o f  the 70S ribosome take p a r t i n the n o n s p e c i f i c b i n d i n g .  The  i n t e g r a t i o n o f p e p t i d y l - t r a n s f e r a s e w i t h i n the s t r u c t u r e o f the 50S r i b o s o m a l s u b u n i t binding  implies  the o c c u r r e n c e o f two tRNA-  s i t e s on the 50S p a r t i c l e a c c e s s i b l e t o a l l tRNA  molecules  (160) .  Further  support comes from the o b s e r v a t i o n  t h a t a n t i b i o t i c s which a c t s p e c i f i c a l l y on the 50S r i b o s o m a l subunit  i n h i b i t the b i n d i n g  r e a c t i o n (158) .  Moreover, the  aminoacyl e s t e r bond o f the aa-tRNA bound t o a 70S ribosome i n response t o mRNA i s p r o t e c t e d (161).  from a l k a l i n e h y d r o l y s i s  Because the presence o f the 50S p a r t i c l e i s a b s o l u t e l y  n e c e s s a r y f o r the p r o t e c t i o n o f the e s t e r , the tRNA appears to be i n t i m a t e l y a s s o c i a t e d w i t h the 50S p a r t i c l e . c o n c l u s i o n was drawn from the o b s e r v a t i o n  The same  t h a t aa-tRNA bound  to 70S ribosomes i n the presence o f a template i s r e s i s t a n t to d i g e s t i o n by p a n c r e a t i c RNase; a g a i n ,  the p r o t e c t i o n o c c u r s  20  as a r e s u l t o f the a s s o c i a t i o n o f aa-tRNA w i t h the 50S somal s u b u n i t ribosomal  (162).  For s p e c i f i c b i n d i n g of tRNA to  s u b u n i t s , the adenosine terminus i s not  b u t the b i n d i n g to 70S r e g i o n and  ribosomes i n v o l v e s both  the  the t e r m i n a l adenosine o f tRNA (163) .  ribo30S  important, anticodon I n summary,  the s u c c e s s f u l f o r m a t i o n o f a p e p t i d e bond r e q u i r e s the c o - o p e r a t i v e i n t e r a c t i o n between a l l components t h a t p a r t i c i pate i n t h i s r e a c t i o n  (164).  Studies concerning  the s u b s t r a t e s p e c i f i c i t y a t the  c a t a l y t i c c e n t e r o f the 50S  ribosomal p e p t i d y l t r a n s f e r a s e  s h o u l d make p o s s i b l e a more d e t a i l e d c h a r a c t e r i z a t i o n of tRNAb i n d i n g s i t e s on the ribosome.  I n g e n e r a l , the f u n c t i o n o f  p e p t i d y l t r a n s f e r a s e seems to be  f a v o r e d by s p e c i f i c i t y a t the  a c c e p t o r s i t e toward s u b s t r a t e s w i t h a f r e e a-amino group  and  by the s p e c i f i c i t y a t the donor s i t e toward s u b s t r a t e s w i t h amido group i n t h a t p o s i t i o n  (165).  There are almost no e x p e r i m e n t a l these n o n s p e c i f i c b i n d i n g f o r c e s . o f 70S  an  d a t a on the nature  I t was  found  that  of  treatment  ribosomes w i t h p r o t e o l y t i c enzymes under m i l d c o n d i t i o n s  a b o l i s h e d the b i n d i n g c a p a c i t y o f ribosomes f o r s p e c i f i c (166).  T h e i r c a p a c i t y to b i n d mRNA was  treatment. ribosomal  Although  not a f f e c t e d by  this  i t l e a v e s the q u e s t i o n unanswered whether  proteins p a r t i c i p a t e d i r e c t l y or i n d i r e c t l y  b i n d i n g of tRNA to ribosomes, t h i s r e s u l t n e v e r t h e l e s s evidence  tRNAs  i n the provides  t h a t the s p e c i f i c tRNA-binding r e a c t i o n r e q u i r e s the  s t r u c t u r a l i n t e g r i t y o f the ribosome.  Treatment of ribosomes  21 w i t h p - d i n i t r o f l u o r o b e n z e n e , t h a t causes directed  l o s s o f the mRNA-  tRNA b i n d i n g a c t i v i t y , y i e l d s some weak  indication  t h a t amino-, t h i o l - , p h e n o l - , o r imidazol-groups o f r i b o s o m a l p r o t e i n s are i n v o l v e d , d i r e c t l y o r i n d i r e c t l y , i n the i n t e r a c t i o n o f tRNA w i t h the template-ribosome Furthermore,  complex  (167) .  the r e a c t i o n o f ribosomes w i t h d i e t h a n o l d i s u l f i d e  d e s t r o y e d the aa-tRNA b i n d i n g a c t i v i t y , whereas the a s s o c i a t i o n o f mRNA w i t h ribosomes normally  (168).  m o d i f i e d i n such a way proceeded  The l o s s o f b i n d i n g a c t i v i t y may be due t o  the f a c t t h a t f r e e t h i o l - g r o u p s a r e necessary t o m a i n t a i n t h e p r o p e r c o n f i g u r a t i o n o f the r i b o s o m a l b i n d i n g s i t e o r t h a t they p a r t i c i p a t e d i r e c t l y  i n the b i n d i n g r e a c t i o n .  b i n d i n g o f N - a c y l a t e d aa-tRNA o r peptidyl-tRNA  S i n c e the  i s not a f f e c t e d  by s u l f h y d r y l - r e a g e n t s b u t o n l y the b i n d i n g o f aa-tRNA, i t was concluded, f i r s t l y ,  t h a t the b i n d i n g s i t e f o r p e p t i d y l -  tRNA i s d i s t i n c t from t h a t f o r aa-tRNA and, s e c o n d l y , t h a t only the acceptor s i t e 30S  i s a l t e r e d by s u l f h y d r y l r e a g e n t s . The  r i b o s o m a l s u b u n i t i s the major s i t e o f i n a c t i v a t i o n  (169).  S u l f h y d r y l reagents do n o t i n h i b i t the f o r m a t i o n o f p o l y p h e n y l a l a n y l - p u r o m y c i n o r o f formylmethionyl-puromycin c a t a l y s e d by 50S r i b o s o m a l s u b u n i t s  (160).  The o c c u r r e n c e o f  a p e n t a n u c l e o t i d e o f c o n s t a n t sequence i n a s i n g l e - s t r a n d e d loop o f the c l o v e r l e a f model o f a l l tRNA-species  a n a l y z e d so  f a r and the e x i s t e n c e o f a complementary sequence i n 5S RNA suggests a d i r e c t i n t e r a c t i o n between tRNA and 50S r i b o s o m a l 5S RNA, mediated  by hydrogen bonds  (258-259).  22  (c) E n z y m e - S p e c i f i c B i n d i n g T h i s w i l l be "The  discussed  of Aminoacyl-tRNA i n the f o l l o w i n g s e c t i o n  Ribosome and P r o t e i n S y n t h e s i s  titled  (see pages 24-28 ) .  (d) Summary There are s i t e ( s ) on the ribosomes which b i n d tRNA. a c t u a l number of s i t e s i s unknown and may experimental c o n d i t i o n s .  For  depend on  i n s t a n c e , Warner and  The  the Rich  (260)  u s i n g i n t a c t r a b b i t r e t i c u l o c y t e s came to the c o n c l u s i o n each ribosome a c t i v e i n p r o t e i n s y n t h e s i s has o f tRNA a t t a c h e d  t o i t . By  molecules  c o n t r a s t , the i n a c t i v e s i n g l e  ribosomes bound approximately one b i n d i n g was  two  molecule of tRNA, and  this  l e s s f i r m than t h a t seen i n the polysomes.  t h e i r extensive  that  In  study of i n v i t r o b i n d i n g of tRNA to  coli  ribosomes, Cannon e t al.(148) found t h a t washed ribosomes bound one  molecule o f tRNA per  exchangeable a t 4 ° . synthesis,  ribosome and  t h i s was  Under c o n d i t i o n s o f a l l - f r e e  the same amount o f tRNA became attached  ribosomes, and  a p o r t i o n o f t h i s was  the o t h e r hand, Takanami become a t t a c h e d  (252,  261)  had  incubation.  r e t i c u l o c y t e ribosomes t h e r e  protein to  the  more f i r m l y bound.  i n the  presence  With polysomal  i s l i t t l e or no exchange a t 4 ° ,  w h i l e i n a c t i v e ribosomes have a l i m i t e d exchange (260). i s p o s s i b l e t h a t some o f these d i f f e r e n c e s are due various  species  i n many r e s p e c t s  involved.  On  found t h a t tRNA w i l l  to r a t l i v e r ribosomes o n l y  o f a t r a n s f e r enzyme d u r i n g  rapidly  The  to  It  the  i n v i t r o environment d i f f e r s  from the environment i n v i v o .  In p a r t i c u l a r ,  23  the r a t e o f p r o t e i n s y n t h e s i s it  i s so much lower i n v i t r o  that  i s not c l e a r t h a t the d i f f e r e n c e between the amount o f tRNA  bound by a c t i v e and i n a c t i v e ribosomes would have been i n the i n v i t r o According  detected  studies. to Wettstein  and N o l l  (262)  ribosomes engaged  i n p r o t e i n s y n t h e s i s b i n d a t l e a s t two and a t most three molecules and t h a t the tRNA bound t o r a t l i v e r  tRNA  polyribosomes  o c c u r s i n t h r e e d i f f e r e n t s t a t e s which e x h i b i t d i f f e r e n t binding properties. peptide-linked  Both the f r e e aminoacyl-changed and the  tRNA are t i g h t l y bound and not removable by  washing, even a t low magnesium, as long as the s t r u c t u r a l i n t e g r i t y o f the a c t i v e complex i s p r e s e r v e d . attachment i s i r r e v e r s i b l e and r e q u i r e s w e l l as energy.  Moreover, t h e i r  t r a n s f e r enzymes as  These three d i f f e r e n t s t a t e s i n which  ribosome-bound tRNA i s encountered correspond t o a t l e a s t two, and p r o b a b l y t h r e e , d i s t i n c t s i t e s on the a c t i v e complex. The f i r s t s i t e , decoding s i t e , s e l e c t s the charged tRNA matching the s p e c i f i e d codon.  S i n g l e ribosomes devoid  have t h i s s e l e c t i v e b i n d i n g  site.  o f mRNA do not  The second s i t e ,  s i t e , which i s found on the 50S r i b o s o m a l s u b u n i t  can  tRNA i n the absence o f mRNA; however, d u r i n g p r o t e i n t h i s s i t e i s only first site. phenylalanine  bind synthesis  a c c e s s i b l e from the a c t i v a t e d s t a t e o f the  G i l b e r t has  shown t h a t tRNA-linked nascent  remains attached  poly-  t o the 50S p a r t i c l e even a f t e r  complete d i s s o c i a t i o n o f the ribosomes i n t o subunits Elson  condensing  (263) .  (264-265) , on the other hand, observed the r e l e a s e o f  24  4S RNA from the 50S p a r t i c l e d u r i n g  i t s conversion  to a p a r t i c l e  w i t h a lower s e d i m e n t a t i o n c o e f f i c i e n t i n the presence o f h i g h salt.  The t h i r d s i t e , e x i t s i t e ,  tRNA.  Not a l l o f the t h r e e  protein synthesis  in vitro.  i s s p e c i f i c f o r uncharged  s i t e s are e q u a l l y o c c u p i e d  during  During p r o t e i n synthesis  i n vivo,  the decoding and condensing s i t e s are both f u l l y  occupied;  however, under i n v i t r o c o n d i t i o n s , a l l o f the condensing and e x i t s i t e s , b u t somewhat l e s s than h a l f o f the decoding are  f i l l e d a t any one moment.  sites  T h i s would i n d i c a t e t h a t t h e  r a t e - l i m i t i n g step i n v i t r o i s the s e l e c t i o n o f tRNA, and i n v i v o i t i s the f o r m a t i o n The  o f the p e p t i d e  Ribosome and P r o t e i n  bond.  Synthesis  Although ribosomes may have s e v e r a l o t h e r  functions i n  v i v o , f o r example, s t i m u l a t i o n o f RNA s y n t h e s i s , o r r e g u l a t i o n o f the b i o s y n t h e s i s o f RNA o r o f ribosomes themselves,  their  o n l y c l e a r l y e s t a b l i s h e d f u n c t i o n s are those r e l a t e d t o the synthesis  of proteins  (70)  and t h i s i s d i s c u s s e d  I n p r o k a r y o t e s the i n i t i a t i o n o f p r o t e i n requires the formation the  (9-11).  synthesis  o f an i n i t i a t i o n complex c o n s i s t i n g o f  30S r i b o s o m a l s u b u n i t ,  (fMet-tRNA^)  below.  mRNA and formyl  methionyl-tRNA  I n a s o l u t i o n o f low M g  + +  ion  t i o n i n i t i a t i o n f a c t o r s as w e l l as GTP are r e q u i r e d step.  concentrafor this  I n i t i a t i o n f a c t o r s are p r o t e i n s which were o r i g i n a l l y  obtained  from crude ribosomes by washing w i t h IM NH^Cl and  were found t o be r e q u i r e d (12-14).  A t l e a s t three  f o r the t r a n s l a t i o n o f n a t u r a l mRNA i n i t i a t i o n f a c t o r s , F i , F 2 and F 3  25 ( a l s o c a l l e d A, B and C r e s p e c t i v e l y ) are known (15. 1 6 ) . The possibility  o f the presence o f a new i n i t i a t i o n  i s now undergoing i n v e s t i g a t i o n .  factor,  I n v i v o the i n i t i a t i o n  site  on the n a t u r a l mRNA c o n t a i n s an AUG codon which codes f o r fMet-tRNA  f  (17-19).  codons i n v i t r o . the  initiation  The codons AUG and GUG serve as i n i t i a t o r  The presence o f a tRNA i n v o l v e d s o l e l y i n  o f p r o t e i n s y n t h e s i s i n b a c t e r i a l systems was  d i s c o v e r e d by Marcker and Sanger appears t o i n i t i a t e  (71).  T h i s tRNA so f a r  s y n t h e s i s o f a l l b a c t e r i a l p r o t e i n s (72-  75) and p r o b a b l y a l l p r o t e i n s i n m i t o c h o n d r i a  (76, 77),  chloroplasts  Preliminary  (78) and b l u e - g r e e n algae  (79).  experiments i n d i c a t e t h a t i n some mammalian systems the mRNA codon assignments f o r p e p t i d e c h a i n i n i t i a t i o n to b a c t e r i a l systems  (235-239, 245) .  are i d e n t i c a l  c o l i has two methio-  Met Met n i n e a c c e p t i n g tRNAs : tRNA* and tRNA . These are both t m changed by the same methionyl-tRNA s y n t h e t a s e  (80) b u t o n l y  Met methionine a t t a c h e d t o tRNA^ formylase  can be f o r m y l a t e d by a t r a n s -  (17) which has been p u r i f i e d  from  c o l i (81).  V e r y r e c e n t l y t r a n s f o r m y l a s e s from wheat germ c h l o r o p l a s t s (229) and from Saccharomyces have been i s o l a t e d .  A c c o r d i n g t o Ochoa  t i o n o f the i n i t i a t i o n Fi and to  c e r e v i s a e m i t o c h o n d r i a (230) (117-118), the forma-  complex i n v o l v e s two steps  : (a)  the  and F3-dependent b i n d i n g o f n a t u r a l mRNA to the ribosomes, (b) the GTP r e q u i r i n g F - d e p e n d e n t b i n d i n g o f fMet-tRNA^ 2  the mRNA-ribosome complex.  initiation  A f t e r f o r m a t i o n o f the  complex c o n s i s t i n g o f the 30S r i b o s o m a l  subunit,  26  mRNA, fMet-tRNA and  the i n i t i a t i o n f a c t o r s , the 50S  s u b u n i t j o i n s t o form the 70S Although  subunit) the 50S  and  subunit).  The  and  30S  t r a n s l o c a t e d to the P s i t e  i s c l e a v e d b e f o r e or d u r i n g  (on  this  82).  T h i s b i n d i n g i s d i r e c t e d by the codon next  r e q u i r e s GTP  w i t h GTP  (on the  next step i s the b i n d i n g o f a second aminoacyl-tRNA  Ts and Tu  of  (25,  the A s i t e .  AUG  GTP  24).  unclear, studies indicate that  a t t a c h e d to the A s i t e  i s subsequently  translocation  to  i n i t i a t i o n complex (23,  the d e t a i l s are s t i l l  fMet-tRNA^ i s i n i t i a l l y  ribosomal  (26).  as w e l l as two  The T f a c t o r s  to  soluble protein factors,  (Ts and Tu)  interact  first  and then w i t h an aminoacyl-tRNA, w i t h the e x c e p t i o n  fMet-tRNA  f  and Met-tRNA  f  (27).  The  GTP-aminoacyl-tRNA-T  f a c t o r complex then r e a c t s w i t h the ribosome l e a d i n g to b i n d i n g of  the aminoacyl-tRNA a t the A s i t e .  hydrolyzed at t h i s step The  GTP  appears to be  (28).  next s t e p i n p r o t e i n s y n t h e s i s i s the f o r m a t i o n o f a  p e p t i d e bond between fMet-tRNA^ (or peptidyl-tRNA) second aminoacyl-tRNA bound t o the ribosome. bond f o r m a t i o n does not r e q u i r e any and  and  the  This peptide  supernatant  protein factor,  i s c a t a l y z e d by p e p t i d y l t r a n s f e r a s e , an enzyme p r e s e n t  on the 50S  ribosomal subunit  (30) .  The p e p t i d e bond  formation  o c c u r s by t r a n s f e r of the a c y l group a t the P s i t e to the amino group o f the aminoacyl-tRNA a t the A s i t e . for  energy  p e p t i d e bond f o r m a t i o n i s s u p p l i e d by the r e l a t i v e l y  energy e s t e r bond between the tRNA and (83).  The  high  the p e p t i d y l moiety  A f t e r f o r m a t i o n o f the f i r s t d i p e p t i d e bond, the  fMet  27 aminoacyl-tRNA stays a t the A s i t e , and the d i s c h a r g e d s t a y s on the ribosome, probably The  tRNA^  a t the o r i g i n a l P s i t e .  next s t e p , t r a n s l o c a t i o n , i n v o l v e s movement o f fMet  aminoacyl-tRNA  (or peptidy1-tRNA) from the A s i t e t o the P  site.  o f discharged  Release  t h i s t r a n s l o c a t i o n step soluble protein factor  tRNA^ from the P s i t e  accompanies  (31). T r a n s l o c a t i o n requires a (G f a c t o r ) and GTP which i s h y d r o l y z e d  to GDP and P i ( 3 2 ) . F a c t o r s s i m i l a r t o T and G and which f u n c t i o n i n the same manner have been i s o l a t e d from mammalian systems (238-242) and y e a s t  (243) .  Simultaneously  w i t h the  t r a n s l o c a t i o n , the ribosome moves along the mRNA, i n the 5'to 3 ' - d i r e c t i o n , by the l e n g t h o f one codon, l e a v i n g t h e t h i r d codon ready the A s i t e .  f o r the b i n d i n g o f a new aminoacyl-tRNA t o  These p r o c e s s e s  are repeated  and p o l y p e p t i d e  c h a i n e l o n g a t i o n c o n t i n u e s u n t i l the ribosome encounters one o f the c h a i n t e r m i n a t i o n codons  (UAG, UAA and UGA).  Recently  the mRNA codon assignments f o r p e p t i d e c h a i n t e r m i n a t i o n have been found  t o be i d e n t i c a l i n mammalian systems  F o r t e r m i n a t i o n t o take p l a c e , the peptidyl-tRNA the P s i t e  (37, 3 8 ) .  the p o l y p e p t i d e  Chain  (97, 233). must be on  t e r m i n a t i o n l e a d s t o cleavage o f  from the tRNA and the subsequent r e l e a s e o f  t h i s tRNA from the ribosome. S i n c e i n i t i a t i o n o f p r o t e i n s y n t h e s i s takes p l a c e on the 30S r i b o s o m a l  s u b u n i t , the ribosomes a f t e r p r o t e i n s y n t h e s i s  must subsequently (110)  undergo d i s s o c i a t i o n .  isolated a dissociation factor  f r a c t i o n which was found  Subramanian e t a l .  (DF) from the 30S f r a c t i o n  t o carry out t h i s function.  Very  28  r e c e n t l y i t has was  been shown (111-112, 231)  actually i n i t i a t i o n factor F .  Therefore  3  shown to have b o t h RNA activities.  by  b i n d i n g and  ribosomes  F  has  3  (113-114) which d i s s o c i a t e  t i o n complex.  ribosomal  T h i s would imply  ribosome-bound and  a f r e e form.  ribosomal  s u b u n i t j o i n s the 30S that F  3  initia-  r e c y c l e s between a  Other i n v e s t i g a t o r s hold  can  3  remain a s s o c i a t e d when they  tRNA.  At present,  the  (115-116,  I f so, F3 might remain ribosome-bound throughout  e n t i r e c y c l e so l o n g as ribosomes, b e a r i n g F , and  been  subsequently  view t h a t ribosomes d i s s o c i a t e a t c h a i n t e r m i n a t i o n 120).  now  termination  i n t e r a c t i o n w i t h F 3 , r e l e a s e d from 30S  s u b u n i t s when the 50S  factor  ribosome d i s s o c i a t i o n  Some workers b e l i e v e t h a t c h a i n  l i b e r a t e s 70S (113)  t h a t t h i s DF  the  associate  c a r r y aminoacyl- or p e p t i d y l -  the p r e c i s e manner i n which F  3  functions  i n the ribosome c y c l e remains an open q u e s t i o n . Release F a c t o r s and  the Mechanism of  When a r i b o s o m a l terminator  : peptidyl-tRNA  Termination complex reaches a  codon on a mRNA, some mechanism must b r i n g about  h y d r o l y s i s o f the e s t e r bond between the p e p t i d y l and moieties.  tRNA  T h i s would then allow the completed p r o t e i n to be  released. Most o f the i n f o r m a t i o n about the mechanism o f t e r m i n a t i o n was these,  RNA  obtained  i n two  from a mutant R17  assay systems.  or f  2  phage w i t h  codon e a r l y i n the c o a t p r o t e i n gene was In v i t r o ,  chain  I n one a UAG  of  nonsense  used as the messenger.  t h i s messenger d i r e c t s the s y n t h e s i s o f the f r e e  29 (not  tRNA-linked) amino t e r m i n a l hexapeptide o f the c o a t  protein  (75, 84-85).  B r e t s c h e r (84) prepared a c e l l - f r e e  system which i n c l u d e d o n l y those aa-tRNA s p e c i e s needed f o r forming the h e x a p e p t i d e . consequence the  I f a codon i s u n t r a n s l a t a b l e i n  o f the l a c k o f a r e q u i r e d aa-tRNA, t h i s l e a d s t o  stoppage o f p e p t i d e c h a i n p r o p a g a t i o n b u t not t o c h a i n  termination.  The f a c t t h a t , i n the presence o f the mRNA from  a nonsense mutant, c h a i n t e r m i n a t i o n o c c u r r e d even i n the absence o f a l l tRNAs, except the s i x s p e c i e s added, suggested t h a t i f RNA was i n v o l v e d i n c h a i n t e r m i n a t i o n , i t was n o t c o n t a i n e d i n the tRNA f r a c t i o n the  (75, 84, 86-87) o b t a i n e d from  h i g h speed s u p e r n a t a n t from  coli.  C a p e c c h i (75) was  a b l e t o p r e p a r e a s u b s t r a t e f o r s t u d y i n g the mechanism o f t e r m i n a t i o n i n the f o l l o w i n g way.  The f o r m a t i o n o f a hexa-  p e p t i d y l - t R N A as s p e c i f i e d by the R17 RNA was b l o c k e d a t the p e n t a p e p t i d y l - t R N A stage by o m i t t i n g from the i n v i t r o the  amino a c i d coded by codon s i x .  system  The ribosome-mRNA-penta-  p e p t i d y l - t R N A complex was then s e p a r a t e d from the s u p e r n a t a n t f r a c t i o n by c e n t r i f u g a t i o n and the aa-tRNA needed the  t o complete  hexapeptidyl-tRNA was added i n the presence o f GTP.  l a s t amino a c i d was then added  t o the c o a t p r o t e i n  The  fragment.  The r e s u l t i n g hexapeptidyl-tRNA remained a t t a c h e d t o the mRNAribosome complex.  T h i s p r o d u c t made p o s s i b l e the study o f the  unique requirements o f the r e l e a s e s t e p . the  I t was found t h a t  r e l e a s e o f f r e e hexapeptide from t h i s complex depended on  a p r o t e i n component from the h i g h speed s u p e r n a t a n t o f T h i s component was d e s i g n a t e d r e l e a s e f a c t o r  (R f a c t o r )  coli (43).  30 Following  C a p e c c h i ' s l e a d , N i r e n b e r g ' s group has been  very  a c t i v e i n studying  tion  (88-98) .  assay.  the f a c t o r s i n v o l v e d i n c h a i n  termina-  Caskey e t a l . (88) used a d i f f e r e n t t e r m i n a t i o n  fMet-tRNA^ was bound t o ribosomes i n the presence o f  the t r i n u c l e o t i d e AUG t o form an AUG-fMet-tRNA^-ribosome complex.  Then a t e r m i n a t o r  t r i n u c l e o t i d e and crude R f a c t o r  were added and the r e l e a s e o f f r e e fMet was measured.  With  t h i s assay they were a b l e t o r e s o l v e R i n t o 2 components, R  x  and  R  2  ; Ri responded t o UAG and UAA b u t n o t UGA whereas Rj  was a c t i v e w i t h UGA and UAA b u t n o t UAG  (89, 9 1 ) . C a p e c c h i  and K l e i n (107) used a n t i s e r a t o p u r i f i e d R  and R  x  to.test  2  t h e i r r o l e i n r e l e a s e o f completed p r o t e i n s i n a c e l l - f r e e system d i r e c t e d by R17 RNA. these f a c t o r s were r e q u i r e d and  Their r e s u l t s indicated that f o r r e l e a s e o f completed  proteins  t h a t e i t h e r f a c t o r c o u l d promote r e l e a s e o f e i t h e r the  c o a t p r o t e i n o r the r e p l i c a s e ; t h i s i m p l i e d t h a t both c i s t r o n s terminated w i t h UAA s i n c e t h i s was the o n l y one o f the three terminator  codons r e c o g n i z e d  by both Ri and R . 2  N i c h o l s (108)  sequenced the p o r t i o n o f R17 RNA a t the end o f the c o a t c i s t r o n and found two c o n s e c u t i v e  terminator  codons, UAAUAG.  T h i s may mean t h a t , a t l e a s t i n some systems, two are r e q u i r e d t o ensure t h a t r e l e a s e o c c u r r e d T h i s seems most l i k e l y  terminators  between c i s t r o n s .  i n view o f the e x i s t e n c e  s u p p r e s s o r s which allow UGA o r UAG t o be read  o f nonsense  as s p e c i f y i n g  an amino a c i d , i n s t e a d o f s e r v i n g as t e r m i n a t o r  signals.  A t h i r d component, S, has been found to i n c r e a s e of formation  o r s t a b i l i t y o f the t e r m i n a t o r  protein  the r a t e  codon : ribosome :  31  f a c t o r R complex (90, 92).  P e p t i d y l t r a n s f e r a s e may  also  be  i n v o l v e d i n r e l e a s e s i n c e i n h i b i t o r s of t h i s enzyme a l s o i n h i b i t release. allow  The  presence of the t e r m i n a t o r  complex  p e p t i d y l t r a n s f e r a s e to a c t as a h y d r o l a s e  break the e s t e r bond h o l d i n g l a s t tRNA (peptidyl-tRNA)  and  thus  the completed p r o t e i n to  (93, 99).  I t was  may  the  previously  suggested t h a t the non-ribosomal enzyme, N-acylaminoacyl-tRNA hydrolase  may  c a t a l y z e the r e l e a s e r e a c t i o n  (102,  106)  but  t h i s now  seems u n l i k e l y i n view of the r e s u l t s of Caskey e t a l .  (88) who  showed t h a t fMet-tRNA i s c l e a v e d  during  the  release  r e a c t i o n w h i l e i t i s known t h a t f r e e fMet-tRNA i s a very substrate isolated  f o r the h y d r o l a s e . an N - s u b s t i t u t e d  supernatant. one,  They suggested t h a t t h e r e  nascent p e p t i d e  hydrolyze  any  J o s t and Bock  aminoacy1-tRNA h y d r o l a s e  a ribosomal hydrolase,  termination,  Recently  and  chains  may  are two  i n the c e l l  i n d i c a t e t h a t R f a c t o r and  Recently  G o l d s t e i n e t a l . (97,  233)  of  chain  the s u p e r n a t a n t h y d r o l a s e ,  tRNA compete f o r the t r a n s l a t i o n of t e r m i n a t o r 100).  hydrolases,  be i n v o l v e d i n the r e l e a s e  oligopeptidy1-tRNA present  Recent s t u d i e s  (232)  from y e a s t  d u r i n g the normal p r o c e s s of  the o t h e r ,  poor  could sap.  suppressor codons  (96,  have i s o l a t e d an  R f a c t o r from r a b b i t r e t i c u l o c y t e s which, i n the presence o f the fMet-tRNA-AUG-ribosome complex and codon causes the r e l e a s e of fMet.  the UAA  terminator  They a l s o found  that  a n t i b i o t i c s which i n h i b i t e d p e p t i d y l t r a n s f e r a s e a c t i v i t y inhibited  also  release.  Very r e c e n t l y I s h i t s u k a and  K a j i (109)  i s o l a t e d another  32 f a c t o r c a l l e d tRNA r e l e a s e f a c t o r (TR) supernatant  of  coli.  I t was  from the h i g h  found to f a c i l i t a t e  removal of tRNA from ribosomes and was  distinctly  from the G f a c t o r i n t h a t i t d i d not r e q u i r e GTP action.  They p o s t u l a t e t h a t t h i s f a c t o r may  o f the ribosome with The  i s probably  for i t s  : A  last  completed  the a c c e p t o r  bound to the donor  s i t e having  codon-specific chain-termination  b i n d to the a c c e p t o r  the  site  termination  f a c t o r would then  s i t e o f the ribosome r e s u l t i n g i n the  s p l i t t i n g o f the p o l y p e p t i d e  group from the tRNA.  i s l e f t on the donor s i t e o f the ribosome and removed by  different  c h a i n l i n k e d to tRNA through the -COOH t e r m i n a l  group o f the p o l y p e p t i d e  codon.  the  a c t a t the  s t e p i n p r o t e i n s y n t h e s i s i n the f o l l o w i n g way polypeptide  speed  The  tRNA  cannot be  the G f a c t o r which r e l e a s e s tRNA only as a conse-  quence o f t r a n s l o c a t i o n .  The  l a s t tRNA from the ribosome.  TR  f a c t o r would then remove the  This hypothesis  t h a t suggested by V o g e l e t a l . (104)  who  complements  postulated  that  p e p t i d y l t r a n s f e r a s e might c l e a v e the s u b s t r a t e , w i t h  the R  f a c t o r a c t i n g to change the s p e c i f i c i t y o f the p e p t i d y l t r a n s f e r a s e r e a c t i o n , so t h a t nascent p e p t i d e was  now  f e r r e d to water i n s t e a d o f to a m o l e c u l e o f aa-tRNA.  transIn  this  event, the R f a c t o r might i n t e r a c t d i r e c t l y w i t h p e p t i d y l t r a n s f e r a s e , i n which case the f a c t o r c o u l d be p a r t of a multimeric  r e l e a s e enzyme.  looked on  A l t e r n a t i v e l y , the R  f a c t o r might i t s e l f c l e a v e the bond between nascent and  tRNA.  The  as  peptide  f a c t o r has been shown to be unable to do  this  33 when free i n s o l u t i o n (104) but i t might be able to catalyze the reaction when the substrate i s bound to a ribosome. From the foregoing discussion, i t can be seen that the mechanism of the actual release r e a c t i o n — t h e cleavage of the ester bond between nascent protein and tRNA and  the  subsequent release of the tRNA—is s t i l l unknown.  I t remains  to be seen whether the R factors do recognize  termination  the  signals d i r e c t l y or whether other molecules, which i n turn i n t e r a c t with the appropriate involved i n t h i s  process.  Ribosomal Structure and The  species of R f a c t o r s , are  Function  foregoing discussion had i l l u s t r a t e d the complexity  of ribosome structure and some of the known events of i t s function i n protein biosynthesis. recent finding by Lodish  (212)  Related to t h i s i s the  that ribosomes from  coli  i n i t i a t e d synthesis i n v i t r o of a l l three of the proteins coded by phage f , but ribosomes from  stearothermophilus  i n i t i a t e d synthesis of only one.  able to show that  2  He was  the s p e c i f i c i t y of i n i t i a t i o n depended on the source of the 30S  ribosomal subunits  subunits, no e f f e c t .  : the o r i g i n of the 50S  ribosomal  i n i t i a t i o n f a c t o r s , tRNA or supernatant enzymes had Thus the 30S p a r t i c l e selects regions of messengers  as signals for the i n i t i a t i o n of polypeptide  synthesis.  It  appears that the control of t r a n s l a t i o n i s dependent on  the  conformation of mRNA (244) .  Presumably the t e r t i a r y structure  of the ribosome plays a c r u c i a l r o l e .  The work of S t e i t z (18)  34  and H i n d l e y  (19) suggested  t h a t the s i t e on the messenger t o  which t h e 30S p a r t i c l e a t t a c h e d was i d e n t i c a l to the s i t e a t which p r o t e i n s y n t h e s i s was i n i t i a t e d . shown t h a t i n f  2  RNA t h e r e are AUG sequences which can i n i t i a t e  p r o t e i n s y n t h e s i s , b u t a r e prevented from doing so (18, 19, 213-215) . more c o m p l i c a t e d ,  I t has a l s o been  by the RNA s t r u c t u r e  To make the problem even  after infection of  c o l i by  phage  ribosomes b i n d a p p r e c i a b l y o n l y t o the m a t u r a t i o n p r o t e i n i n i t i a t i o n s i t e o f R17 RNA i n the i n i t i a t o r  factor  (216) .  Apparently  t h i s change  lies  fraction.  R e c e n t l y i n o r d e r t o g e t a c l e a r e r p i c t u r e o f the ribosome, s t u d i e s on the low m o l e c u l a r weight RNA bound t o ribosomes have been s t a r t e d by many groups w i t h the hope o f b e i n g a b l e t o c o r r e l a t e the f i n d i n g s w i t h a p o s s i b l e c l e a r e r understanding  o f the s t r u c t u r e o f the ribosome w i t h r e s p e c t  to i t s f u n c t i o n . U n t i l r e c e n t l y RNA was c o n s i d e r e d to belong  t o one o f  those c a t e g o r i e s : t r a n s f e r , r i b o s o m a l o r messenger.  Develop-  ment o f more s o p h i s t i c a t e d a n a l y t i c a l t e c h n i q u e s , such as p o l y a c r y l a m i d e g e l e l e c t r o p h o r e s i s and improved  chromatographic  t e c h n i q u e s , has p e r m i t t e d the d e t e c t i o n o f a number o f new RNA s p e c i e s , among which are RNAs o f r e l a t i v e l y weight  (5-8S) .  Such s m a l l RNAs have been found i n o r  a s s o c i a t e d w i t h ribosomes  (170, 175), microsomal membrane  smooth endoplasmic r e t i c u l u m (186), n u c l e i 191,  low m o l e c u l a r  194) and n u c l e o l i  (185),  (181, 182, 187, 188,  (188, 190, 193).  K n i g h t and Sugiyama  (170) found  a new c l a s s o f tRNA i n  35  E. c o l i  ribosomes and  supernatant  but not i n HeLa c e l l cytoplasm. associated with a molecular  ribosomes.  and  i n HeLa c e l l  mitochondria  Most o f the tRNA i s not  T h i s minor tRNA was  found to have  weight, judged by m o b i l i t y on p o l y a c r y l a m i d e  intermediate  between the major p a r t of tRNA and  minor tRNA of E ^ c o l i  gels,  5S RNA.  The  can be changed w i t h amino a c i d s , however,  i t s amino a c i d s p e c i f i c i t y i s d i f f e r e n t from t h a t of the major c l a s s o f tRNA. A f u r t h e r study  by K n i g h t  (171)  revealed that  ethidium  bromide treatment o f growing HeLa c e l l s e l i m i n a t e d t h i s minor tRNA from the m i t o c h o n d r i a  and  reduced the major s p e c i e s  50% w i t h o u t  a f f e c t i n g the s y n t h e s i s o f 5S RNA.  s p e c i e s was  found to be unmethylated w h i l e  had  a completely  cytoplasmic  d i f f e r e n t methylation  4S RNA.  between the 5S RNA mitochondrial  No  Recently  and  from the m i t o c h o n d r i a  precursor  I t has  tRNA (183,  173).  184) .  This  a l s o been shown t h a t the tRNA  of r a t l i v e r and Neurospora have  The m i t o c h o n d r i a l  tRNA was  efficiently  to the m i t o c h o n d r i a l DNA  cytoplasmic  tRNA s u g g e s t i n g  counterparts  found to h y b r i d i z e more  than the  that mitochondrial  t r a n s c r i b e d from m i t o c h o n d r i a l DNA. Nass and Buck (174)  (pre-tRNA)  on g e l e l e c t r o p h o r e s i s  d i f f e r e n t s p e c i f i c i t e s from t h e i r c y t o p l a s m i c (172,  the  t h a t o c c u r r i n g i n the  pre-tRNA which i s not methylated migrated 5S RNA.  the major s p e c i e s  p a t t e r n from  has been i s o l a t e d from HeLa c e l l cytoplasm  between tRNA and  minor  s i g n i f i c a n t d i f f e r e n c e s were observed  o f the cytoplasm  fraction.  The  by  corresponding tRNA i s  In a recent report,  observed t h a t the f o u r m i t o c h o n d r i a l  tRNAs  36  they s t u d i e d from r a t l i v e r d i f f e r e d i n base sequences t h e i r cytoplasmic counterparts.  from  These m i t o c h o n d r i a l tRNAs  c o u l d o n l y be a c y l a t e d by m i t o c h o n d r i a l synthetases and  not  by c y t o p l a s m i c enzymes. R e c e n t l y 7S RNA somes (175). rRNA and 28S  rRNA.  I t was  as such may  has been i s o l a t e d from HeLa c e l l found t o be a s s o c i a t e d w i t h the  ribo28S  p l a y a r o l e i n the conformation o f the  They a l s o p r e s e n t evidence  t h a t 7S RNA  derives  from the same p o l y n u c l e o t i d e p r e c u r s o r as does i t s accompanying 28S molecule  (175,  217) .  7S RNA  was  hamster and c h i c k e n f i b r o b l a s t 28S to be one  7S RNA  molecule  a l s o found i n Chinese  rRNA (175).  There  f o r every 28S molecule  appears  and i t appears  to be a t t a c h e d to the l a r g e r rRNA by non-covalent bonds. 7S c o n t a i n s approximately  one methylated  m o b i l i t y of 4, 5 and  7S RNA  between 4 and  and  5S RNA  5 and 7S  RNA.  have observed  low m o l e c u l a r weight s p e c i e s of RNA  s i z e from 4 to 6S RNA, w i t h the n u c l e u s .  Electrophoretic  r e v e a l s about an equal s e p a r a t i o n  Weinberg and Penman (181)  n u c l e o l u s o f HeLa c e l l s .  base.  The  six distinct  i n the nucleoplasm  n u c l e a r RNA  s p e c i e s range i n  and appear to be s t a b l y a s s o c i a t e d  n u c l e o p l a s m i c and o t h e r s w i t h the n u c l e o l a r f r a c t i o n . and have base compositions  o f the o t h e r n u c l e a r RNA o n l y about 0.4% 5S RNA  and  Some are a s s o c i a t e d p r i n c i p a l l y w i t h  s p e c i e s are methylated  The  classes.  These  u n l i k e any  These s p e c i e s r e p r e s e n t  of the t o t a l c e l l u l a r RNA.  i s p r e s e n t i n g r e a t excess  the  I n the n u c l e u s ,  r e l a t i v e to 7S RNA  (175;  37  t h i s RNA e x h i b i t s both  the s e d i m e n t a t i o n  v e l o c i t y and e l e c t r o -  p h o r e t i c m o b i l i t y o f a s p e c i e s o f approximately  5.5S  RNA,  the term 7S RNA o r i g i n a l l y g i v e n to t h i s s p e c i e s by Pere e t a l . i s thus a misnomer) which i s c o n s i s t e n t w i t h the e x i s t e n c e o f a s i z e a b l e p o o l o f 5S ribosomal RNA i n the nucleus.  The 5.5S and 5S RNA s p e c i e s are p r e s e n t i n  equimolar  q u a n t i t i e s i n c y t o p l a s m i c ribosomes.  s p e c i e s o f n u c l e a r RNA a r e found and  i n the d e v e l o p i n g  Similar  i n mouse f i b r o b l a s t c u l t u r e s  c h i c k embryo b r a i n .  Preparative g e l  e l e c t r o p h o r e s i s has been used t o i s o l a t e four methylated m o l e c u l a r weight RNA molecules (187). and  from Chinese hamster  low  cells  They have been shown to be d i s t i n c t from tRNA, 5S  5.5S rRNA and are found  respect to n u c l e i .  i n a f r a c t i o n enriched  with  I n a more r e c e n t i n v e s t i g a t i o n , Weinberg  and Penman have i s o l a t e d t h r e e new n u c l e a r low m o l e c u l a r RNA s p e c i e s  (182) .  One s p e c i e s migrates  with  weight  identical  e l e c t r o p h o r e t i c m o b i l i t y to t h a t o f 5S rRNA and i s methylated while  the o t h e r two s p e c i e s , a l s o methylated,  than 5.5S RNA.  These RNAs are found  migrate  slower  almost s o l e l y i n the  n u c l e i o f i n t e r p h a s e c e l l s and a r e q u i t e l o o s e l y a s s o c i a t e d with  the n u c l e o p r o t e i n complexes o f the nucleus which i s i n  marked c o n t r a s t t o the o t h e r p r e v i o u s l y d e s c r i b e d s i x s p e c i e s of n u c l e a r RNA.  A l l nine n u c l e a r RNA s p e c i e s d i f f e r  from each  o t h e r i n s e v e r a l o f t h e i r p r o p e r t i e s ; f o r example, s t a b i l i t y , s i z e , s y n t h e s i s , q u a n t i t y , e t c . , which may r e f l e c t d i f f e r e n c e s i n their function i n vivo. Busch's group has shown t h a t the n u c l e i and n u c l e o l i o f  38  normal r a t l i v e r  (188)  and a number of tumor c e l l s  c o n s i s t e n t l y c o n t a i n e d 4 to 6S RNA. 6S RNA and  has been i s o l a t e d  (189)  R e c e n t l y n u c l e o l a r 4 to  from N o v i k o f f hepatoma a s c i t e s  cells  s e p a r a t e d i n t o t h r e e main f r a c t i o n s by e x c l u s i o n chromato-  graphy on Sephadex G-100  (190).  The e l u t i o n p a t t e r n was  to be s i m i l a r to the r i b o s o m a l 4 to 6S RNA hepatoma.  of the N o v i k o f f  Peak I (the f r a c t i o n t h a t emerged i n the v o i d  volume) c o n t a i n d two major components, both of lower than r i b o s o m a l 5S RNA.  Only one band migrated  a c r y l a m i d e g e l s and was  s i m i l a r to the RNA  ribosomes.  mobility  i n 10%  and Dingman (191) weight RNA  4S RNA  r e p o r t e d the presence  s p e c i e s (4 to 7S RNA)  d e r i v e d from  respectively. of s e v e r a l low  Hodett  and Busch  t h i s n u c l e a r f r a c t i o n and c h a r a c t e r i z e d two U - r i c h RNA (192,  195,  l e s s than 5S RNA,  197).  molecular  isolated frac-  These U - r i c h f r a c t i o n s , w i t h m o b i l i t y  are a p p a r e n t l y u n r e l a t e d to those found  o t h e r workers f o r HeLa c e l l ribosomes (185).  Peacock  i n r a t l i v e r n u c l e i which  were not p r e s e n t i n the cytoplasm.  fractions  poly-  The main component i n Peaks I I and I I I migrated  w i t h m o b i l i t i e s s i m i l a r to 5S and  tions  found  (175)  and  by  cytoplasmic  Each of these o t h e r f r a c t i o n s seem to be  p r e s e n t p r i m a r i l y i n the rRNA or r i b o s o m a l s u b f r a c t i o n s . Busch suggests  t h a t s i n c e t h i s U - r i c h RNA  l a b e l i n g than any o t h e r n u c l e a r RNA,  has a lower r a t e of  t h i s RNA  would appear to  be s t a b l e and might serve a s t r u c t u r a l r o l e but s i n c e t h i s  RNA  i s l e s s hydrogen-bonded than tRNA o r rRNA i t might a l s o e x e r t some r o l e i n template  activity.  R e c e n t l y two o t h e r low mole-  c u l a r weight RNAs have been l i b e r a t e d from the n u c l e o l a r 28S  39  RNA f r a c t i o n and r e f e r r e d t o as 8S and U3 RNA  (193) .  two RNAs a r e n o t a s s o c i a t e d w i t h r i b o s o m a l 28S RNA.  These The  molar r a t i o o f 8S and U3 RNA to n u c l e o l a r 28S RNA i s o n l y approximately  1:2, s u g g e s t i n g t h a t o n l y some o f the n u c l e o l a r  28S RNAs a r e bound to these m o l e c u l e s .  They may have some  r o l e i n the f o r m a t i o n o f the u l t i m a t e c y t o p l a s m i c r i b o s o m a l particle.  I t i s p o s s i b l e t h a t they may a l s o serve as e s s e n t i a l  components  f o r the movement o f n u c l e o l a r products  to the  n u c l e a r r i b o n u c l e o p r o t e i n network, o r f o r the a d d i t i o n o f s p e c i a l p r o t e i n s t h a t are components o f the ribosomes. n u c l e a r RNA has a l s o been i s o l a t e d from KB c e l l s  An 8S  (246) .  More  r e c e n t l y U3 RNA and 4.5S RNA have been i s o l a t e d from the nucleus o f N o v i k o f f hepatoma c e l l s and from r a t l i v e r (194-195).  T h i s 4.5S RNA was subsequently  three f r a c t i o n s .  into  I t s h o u l d be noted t h a t the 3 ' - t e r m i n a l o f  one f r a c t i o n i s b l o c k e d  (196).  R e c e n t l y the n u c l e o l a r U3 RNA  was s e p a r a t e d i n t o 4 d i s t i n c t bands suggested  separated  nuclei  (198) .  I t has been  t h a t these RNAs may f u n c t i o n i n p r o c e s s i n g o f  n u c l e o l a r r i b o s o m a l RNA p r e c u r s o r s i n t o r i b o s o m a l p r e c u r s o r p a r t i c l e s and f i n a l l y o f these molecules  i n t o mature ribosomes.  suggests  The low t u r n o v e r  t h a t some might be s t a b l e messenger  RNAs f o r c e r t a i n p r o t e i n s , perhaps r i b o s o m a l o r r i b o s o m a l precursor proteins.  I t i s a l s o p o s s i b l e t h a t these RNAs may  p l a y a r o l e as i n i t i a t i o n f a c t o r s i n ribosomal RNA s y n t h e s i s . V a r i o u s RNAs have been i s o l a t e d from r a b b i t b e s i d e s 4, 5 and 7S RNA  (207-208).  8S and 10S have p r o p e r t i e s expected  reticulocytes  Two RNAs sedimenting a t o f mRNA.  40  Sea u r c h i n 26S bonded 5.8S  rRNA  rRNA has been shown to c o n t a i n a  (199), which i s unmethylated  s i m i l a r to the 5.5S  RNA  5.5S  Sy and McCarty  and which i s  a s s o c i a t e d w i t h rRNA of HeLa c e l l s  (175) , c h i c k e n f i b r o b l a s t s (193) .  hydrogen-  (175)  suggest  and N o v i k o f f a s c i t e s  (199)  t h a t the 5.8S  rRNA i s i n v o l v e d i n m a i n t a i n i n g the c o r r e c t  tumors  rRNA or the 3-dimensional  c o n f i g u r a t i o n o f e i t h e r the 28S or 26S rRNA necessary f o r i t s p r o p e r i n t e r a c t i o n w i t h p r o t e i n s i n ribosome Y e a s t ribosomes and 5S RNA,  a 5.8S  have been found to c o n t a i n b e s i d e s 4S  RNA  molecule which i s unmethylated  which i s n o n - c o v a l e n t l y a t t a c h e d t o the 25S i s suggested t h a t the 5.8S 35S p r e c u r s o r RNA,  maturation.  RNA  rRNA  and  (200) .  It  i s d e r i v e d from a p a r t of the  whereas the 5S RNA  i s made de novo.  I t has been found, j u s t r e c e n t l y , t h a t the m i t o c h o n d r i a l ribosomes A 7S RNA  of Neurospora component was  c r a s s a are d e v o i d of 5S RNA also  (201) .  absent.  A f t e r i n f e c t i o n w i t h adenovirus 2 o r 7, human e p i t h e l i o i d cells RNA of  s y n t h e s i z e a d i s c r e t e s p e c i e s of low m o l e c u l a r weight  (176).  T h i s i s found predominantly  the cytoplasm of these c e l l s .  i n the s o l u b l e  fraction  I t s f u n c t i o n i s unknown,  and i t s primary s t r u c t u r e i s d i f f e r e n t from t r a n s f e r RNA from the two p r i n c i p a l low m o l e c u l a r weight RNA found i n the ribosomes m o l e c u l a r weight RNA predominantly  3 to 4 mins. a f t e r complete  T h i s type o f RNA  components  o f the u n i n f e c t e d KB c e l l s  synthesis i n T5-infected  s y n t h e s i s was  bands of m o l e c u l a r weight range  (177).  t o 3.1  Low  c o l i occurred  infection  (178).  c h a r a c t e r i z e d by seven 8.0  and  discrete  x 10"* and a broad  41 band m i g r a t i n g e q u i v a l e n t t o h o s t 4S RNA.  The d a t a  suggested  t h a t a l l s p e c i e s o f m o l e c u l a r weight between 5.3 and 2.6 x 10  k  were p r o b a b l y cleavage products o f RNA o f h i g h e r m o l e c u l a r weight.  Only one band, m o l e c u l a r weight,  shown to be bound t o polysomes. p r e c u r s o r s from  coli  5.3 x 10" has been  Altman has i s o l a t e d  tRNA  i n f e c t e d w i t h b a c t e r i o p h a g e <J>80 (205) .  The p r e c u r s o r migrated on p o l y a c r y l a m i d e g e l s between 4S and 5S RNA.  Many s p e c i e s o f low m o l e c u l a r weight RNA r a n g i n g  from 4S to 10S have been i s o l a t e d  from Rous Sarcoma V i r u s  (179, 180). P u r i f i e d RSV c o n t a i n a homogeneous p o p u l a t i o n o f methylated  4S RNA which i s i n d i s t i n g u i s h a b l e from h o s t tRNA  on the b a s i s o f e l e c t r o p h o r e t i c m o b i l i t y , although i n n u c l e o t i d e composition are d e t e c t a b l e .  differences  A minor homogeneous  RNA component, w i t h s e d i m e n t a t i o n v e l o c i t y and e l e c t r o p h o r e t i c m o b i l i t y approximating  the 7S RNA molecule, and a few methyl  r e s i d u e s has been d e t e c t e d .  I t s s i g n i f i c a n c e and p o s s i b l e  f u n c t i o n are p r e s e n t l y o b s c u r e . R e c e n t l y the n u c l e o t i d e sequence o f 6S RNA from the supernatant f r a c t i o n of  coli  has been e s t a b l i s h e d  (202) .  T h i s RNA had been noted p r e v i o u s l y although no f u n c t i o n has been a s s i g n e d t o i t (203-204) . found i n h i g h e r organisms  I t s r e l a t i o n s h i p w i t h RNAs  i s unclear.  I t may be r e l a t e d to  one o r o t h e r o f s e v e r a l bands of r a t h e r s i m i l a r  electrophoretic  m o b i l i t i e s t o 6S RNA on acrylamide g e l e l e c t r o p h o r e s i s ( b e l i e v e d t o be n u c l e a r RNAs) (191, 197). I t i s , however, u n l i k e l y t o be r e l a t e d to the low m o l e c u l a r weight  ribosomal  42  RNA, c a l l e d 7S RNA on  (175) , f o r 6S RNA o f  coli  i s n o t found  ribosomes. I n summary, a new and e x c i t i n g  c h a p t e r has r e c e n t l y  opened up i n the f i e l d o f RNA chemistry and p a r t i c u l a r l y  that  of the b i o c h e m i s t r y o f n u c l e o l a r , n u c l e a r and rRNA; f o r example, the u n i q u e l y l o c a l i z e d low m o l e c u l a r weight RNA. elucidation of their functions w i l l of  r i b o s o m a l s t r u c t u r e and f u n c t i o n .  The e v e n t u a l  h e l p i n the understanding  43 OUTLINE OF THE PROBLEM The  events i n v o l v e d i n the i n i t i a t i o n o f p r o t e i n  s i s are r e l a t i v e l y w e l l understood.  However, the mechanism o f  chain termination i n protein biosynthesis i s s t i l l problem. but  synthe-  an  unsolved  The d i s c o v e r y o f R f a c t o r s was a g r e a t s t e p  forward,  i n c o n t r a s t t o the case o f i n i t i a t i o n where a s p e c i f i c  tRNA i s d e f i n i t e l y  i n v o l v e d , the requirement f o r a s p e c i f i c  tRNA i n t e r m i n a t i o n has n o t , as y e t , been shown and the p r e s e n t understanding required. previous t i o n , p.  o f the problem i s t h a t such a tRNA i s n o t  The c h a i n - t e r m i n a t i n g  experiments o u t l i n e d i n a  s e c t i o n (Release F a c t o r s and the Mechanism o f Termina28  ) i n which h i g h l y p u r i f i e d  tRNAs were used,  n e v e r t h e l e s s were not p r o p e r l y c o n t r o l l e d s i n c e i t can be argued t h a t the t e r m i n a t i n g ribosomes.  The p r e v i o u s  tRNA c o u l d remain bound t o the  i n v e s t i g a t o r s f a i l e d t o show t h a t  t h e i r ribosomes o r r i b o s o m a l  s u b u n i t s were devoid o f 4S  Perhaps t h i s h y p o t h e t i c a l c h a i n - t e r m i n a t i n g  RNA.  tRNA i s d i f f e r e n t  i n some manner from the normal tRNA such t h a t i t i s n o t removed from the ribosome d u r i n g the c l e a n i n g p r o c e d u r e . any  In  event, i t may combine i n some manner with R f a c t o r , o r  some o t h e r p r o t e i n , such as p e p t i d y l t r a n s f e r a s e i n the t e r m i n a t i o n mechanism, o r i t may simply be p r e s e n t ribosomal  conformation  f o r the t e r m i n a t i o n p r o c e s s .  are o n l y some o f the q u e s t i o n s f i n a l mechanism can be Recently  to s t a b i l i z e These  t h a t must be answered b e f o r e a  hypothesized.  i n v e s t i g a t i o n s have l e d t o c o n s i d e r a b l e under-  s t a n d i n g o f the r i b o s o m a l  structure.  Both subunits have been  r e c o n s t i t u t e d w i t h p a r t i c u l a r emphasis  on the 30S s u b u n i t s  and the o r d e r i n which the p r o t e i n s are reassembled to form the of  functional unit. the RNA  L e s s i s known about the b i o l o g i c a l  a s s o c i a t e d w i t h ribosomes.  role  I n p a r t i c u l a r the  nature and f u n c t i o n o f many s m a l l m o l e c u l a r weight RNAs a s s o c i a t e d w i t h ribosomes i s unknown. Thesis Proposal T h i s t h e s i s was  devoted to the i n v e s t i g a t i o n and c h a r a c -  t e r i z a t i o n o f the low m o l e c u l a r weight RNA  bound to  coli  ribosomes which had been p r e p a r e d by s t a n d a r d p r o c e d u r e s . Methods were a l s o s t u d i e d f o r p r e p a r i n g ribosomes d e v o i d o f all  4S components w i t h p a r t i c u l a r emphasis  on the r o l e o f  tRNA i n the c h a i n t e r m i n a t i o n mechanism. Ribosomes from Ej_ c o l i were chosen f o r study not o n l y because they are so w e l l c h a r a c t e r i z e d and have been used i n p r e v i o u s c h a i n t e r m i n a t i o n s t u d i e s b u t a l s o because o f the ease w i t h which these a c t i v e ribosomes can be o b t a i n e d .  The  e x p e r i m e n t a l o u t l i n e i s : (a) i s o l a t i o n and p r e p a r a t i o n o f a c t i v e ribosomes  (WRIb), (b) removal of bound tRNA from WRib  and subsequent c h a r a c t e r i z a t i o n , the  low m o l e c u l a r weight  s u b u n i t s , and  (d) exchange  RNA  (c) c h a r a c t e r i z a t i o n o f  from whole ribosomes and the  o f l a b e l e d r i b o s o m a l bound tRNA  w i t h u n l a b e l e d tRNA and subsequent  characterization.  45  MATERIALS AND METHODS Chemicals Common c h e m i c a l s o b t a i n e d commercially were o f the h i g h e s t p u r i t y o r reagent grade.  Individual  amino a c i d s i n c l u d i n g the amino a c i d mixture and  3  radioactive ( **C-labelled) 1  H - l a b e l l e d u r a c i l were o b t a i n e d from New England N u c l e a r  C o r p o r a t i o n ; adenosine 5 ' - t r i p h o s p h a t e (disodium) guanosirie 5 ' - t r i p h o s p h a t e (trisodium) polyuridylic acid  (ATP)  and  (GTP) , from Calbiochem;  (poly U), from M i l e s Chemical Company;  puromycin d i h y d r o c h l o r i d e  (PM.2HC1), from N u t r i t i o n a l Biochem-  i c a l s Corporation; N,N -Methylenebisacrylamide 1  N'-Tetramethylethylenediamine  ( B i s ) , N,N,N',-  (TEMED), 2-mercaptoethanol,  r i b o f l a v i n and a u r i n t r i c a r b o x y l i c a c i d  (ATA), from Eastman  O r g a n i c Chemicals; methylene b l u e from F i s h e r S c i e n t i f i c Co.; a c r i d i n e orange  ( b a s i c orange 14), a c r y l a m i d e and 3'-dimethyl-  aminopropionitrile  (DMAPN), from Matheson, Coleman and B e l l ;  lanthanum a c e t a t e from K&K L a b o r a t o r i e s , I n c . ; cesium chloride  ( C s C l ) , A grade f o r d e n s i t y g r a d i e n t s , from C a l b i o -  chem; s u c r o s e , d e n s i t y - g r a d i e n t grade  (ribonuclease-free),  from Mann Research L a b o r a t o r i e s , I n c . ; t r a n s f e r RNA (tRNA) from  c o l i B, from G e n e r a l B i o c h e m i c a l s ; and DNase I from  Worthington B i o c h e m i c a l Corp. Preparation o f E. c o l i B (a)  C e l l s grown t o the l a t e l o g phase were o b t a i n e d from the  G r a i n P r o c e s s i n g Co., Muscatine, Iowa. used was as f o l l o w s  The growth medium  : 1% g l u c o s e , 1% y e a s t e x t r a c t i n a  46 phosphate b u f f e r medium, the b u f f e r s b e i n g monopotassium and d i p o t a s s i u m phosphate.  The s t a r t i n g pH was 7.0 t o 7.1.  the growth p e r i o d was completed, follows  After  the c e l l s were h a r v e s t e d as  : (1) the c o n t e n t s o f the fermentor were c o o l e d  from  37° t o 16° and then c e n t r i f u g e d , (2) the c e l l s were r e c o v e r e d from the c e n t r i f u g e and washed i n a medium made up o f 0.5% KC1 at  and 0.5% NaCl,  (3) the c e l l s were r e c e n t r i f u g e d and f r o z e n  -20°.  (b) One hundred mis o f medium (see below) i n a 125-ml e r l e n meyer f l a s k was i n o c u l a t e d w i t h  c o l i p r e v i o u s l y grown on the  s u r f a c e o f an agar s l a n t and the f l a s k was a e r a t e d by shaking at  100 rpm on a M e t a b o l i t e Water Bath Shaker  Scientific log  Co., Inc.)  s e t a t 37°.  (New Brunswick  When growth had reached mid-  phase, the c o n t e n t s o f the f l a s k , which s e r v e d as the  inoculum, were added t o a carboy c o n t a i n i n g 15 l i t e r s o f the f o l l o w i n g minimal  medium a t pH 7.0 (g/L) :  K HPO KH POi, Na c i t r a t e . 2 H 0 MgSO*.7H 0 NIUC1 glucose 2  2  2  2  7.0 3.0 0.5 0.2 1.0 4.0  autoclaved together f o r 20 mins.  Throughout the growth p e r i o d the c e l l s were v i g o r o u s l y aerated.  When the c e l l s had reached the l a t e l o g phase, they  were q u i c k l y c o o l e d by adding i c e and then h a r v e s t e d by c e n t r i f u g a t i o n and s t o r e d a t - 7 0 ° . P r e p a r a t i o n o f E . c o l i B P y r i m i d i n e - R e q u i r i n g Mutant 12632 (American  Type C u l t u r e C o l l e c t i o n , ATCC 13135)  47  CHART .1* P r e p a r a t i o n o f E. c o l i  ribosomes  E._ c o l i a) ground w i t h g l a s s beads i n b u f f e r A (10 mMMg(0Ac) ) b) c e n t r i f u g e d a t 10,000 x g debris supernatant a) i n c u b a t e d w i t h DNase b) c e n t r i f u g e d 2x a t 30,000 x g 2  cellular  precipitate  1  supernatant c e n t r i f u g e d at 105,000 x g ribosomal p e l l e t supernatant a) mixed w i t h (S100) b u f f e r B (0.1 mM Mg(OAc) ) b) c e n t r i f u g e d a t 10 5,000 x g 2  ribosomal p e l l e t S100A a) mixed w i t h b u f f e r C (1 mM Mg(OAc) ) b) c e n t r i f u g e d a t 105,000 x g 2  ribosomal p e l l e t supernatant a) mixed w i t h b u f f e r D (5 mM Mg(OAc) ) b) c e n t r i f u g e d a t 105,000 x g 2  ribosomal p e l l e t supernatant a) mixed w i t h b u f f e r E (10 mM Mg(OAc) ) b) c e n t r i f u g e d a t 105,000 x g c) r e p e a t e d a) and b) 3x 2  ribosomal p e l l e t supernatant [mixed w i t h b u f f e r F (10 mM Mg(OAc) ) 2  r i b o s o m a l suspension (WRib) a) Put on D E A E - c e l l u l o s e column e q u i l i b r a t e d w i t h b u f f e r G and wash with same b u f f e r b) e l u t e d w i t h b u f f e r H r i b o s o m a l suspension c e n t r i f u g e d a t 105,000 x g "I r ribosomal p e l l e t supernatant I mixed w i t h b u f f e r F 1'  r i b o s o m a l suspension (RSI)  * B u f f e r A-F d e s c r i b e d  p r e v i o u s l y i n L i s t of B u f f e r s .  48 An inoculum was  prepared from the  c o l i mutant grown on  the s u r f a c e o f an agar s l a n t as i n (b) above.  When growth  has reached the mid-log phase, the inoculum was 1 5 - l i t e r carboy c o n t a i n i n g the same medium as t h a t 4 yg u r a c i l / m l medium was were r e q u i r e d ,  3  added.  H - l a b e l l e d u r a c i l was  added to a (b) above except  Where l a b e l l e d  cells  added to the carboy  p r i o r to l o g phase i n presence of the same amount o f u n l a b e l l e d uracil. as  The  c e l l s were grown and h a r v e s t e d i n the same manner  (b) above and f i n a l l y s t o r e d a t - 2 0 ° .  P r e p a r a t i o n o f E . c o l i B Ribosomes  (WRib) (see C h a r t I)  Ribosomes were prepared from E_^ c o l i by a combination of many methods (218-220) which were i n t e n d e d to remove a l l nonribosomal m a t e r i a l .  T h i s was  a c h i e v e d by employing  a series  of b u f f e r s w i t h the same h i g h ammonium c o n c e n t r a t i o n but w i t h v a r y i n g magnesium c o n c e n t r a t i o n s . A l l o p e r a t i o n s were performed  a t 0°-4° .  t h r e e times  F r o z e n o r f r e s h c e l l s were ground w i t h  (3x) the weight o f S u p e r b r i t e g l a s s beads  (3M  Company, p r e v i o u s l y c l e a n e d i n 6NHC1 and washed w i t h d i s t i l l e d water) by means o f a V i r t i s 45 homogenizer running a t top speed i n the presence of an e q u a l volume of b u f f e r A Tris,  10 mM  7.8).  The  Mg(OAc) » 2  lOmM NHi,Cl, 10 mM  (10  mM  mercaptoethanol,  pH  c e l l s were homogenized f o r f o u r 2 minute i n t e r v a l s  w i t h f i v e minute c o o l i n g p e r i o d s i n between. f l a s k was  The  homogenizing  surrounded by i c e throughout these o p e r a t i o n s .  F o l l o w i n g the l a s t homogenization,  the homogenate  was  c e n t r i f u g e d a t 10,000 x g f o r 20 mins i n o r d e r to remove  49  c e l l u l a r debris.  The  3 yg o f p a n c r e a t i c  s u p e r n a t a n t f r a c t i o n was  DNase I was  added w i t h g e n t l e mixing  each m i l l i l i t e r  of  The  e x t r a c t was  then c e n t r i f u g e d  The  s u p e r n a t a n t s o l u t i o n was  repeated. was  c o l l e c t e d and  c o l i e x t r a c t and i n c u b a t e d f o r 10 mins. a t 30,000 x g f o r 20 mins.  removed and the c e n t r i f u g a t i o n  The upper f o u r - f i f t h s o f the supernatant  removed and c e n t r i f u g e d  Model-L u l t r a c e n t r i f u g e .  solution  a t 105,000 x g f o r 5 h r s i n a  The  s u p e r n a t a n t was  removed and  d i a l y z e d v e r s u s b u f f e r A o v e r n i g h t (30:1 r a t i o ) and stored  a t -70° .  T h i s was  l a b e l l e d S-100  then  and i s the source  o f aminoacyl-tRNA s y n t h e t a s e s and f a c t o r s r e q u i r e d synthesis The Tris,  and was  used  i n many subsequent  r i b o s o m a l p e l l e t was  0.1  mM  for 2 hrs.  Mg(OAc) f 2  Aggregates  dissolved  for protein  analyses.  i n buffer B  0.5-1.0 M NHi,Cl, pH 7.4)  (10  were removed by c e n t r i f u g a t i o n a t  a t 105,000 x g f o r 15-24  S1Q0A, was  saved and  initiation  and  The Tris, 2 hrs.  stored  Aggregates  W  hrs.  The  Mg(OAc) , 0.5-1.0 M NtUCl, pH  Aggregates  supernatant,  i s the source o f  for protein i n buffer C 7.4)  synthesis. (10  mM  and mixed f o r  then the r i b o s o m a l mixture was  the r i b o s o m a l p e l l e t d i s s o l v e d 2  was  were removed by c e n t r i f u g a t i o n a t 10,000  a t 105,000 x g f o r 10-15  5 mM  dissolved  Mg(OAC) , 0.5-1.0 M NH C1, pH 2  The  This  transfer factors required  x g f o r 5 mins and  and  hrs.  at -70°.  r i b o s o m a l p e l l e t was  1 mM  mM  and mixed  10,000 x g f o r 5 mins and then the r i b o s o m a l mixture centrifuged  to  s u p e r n a t a n t was i n buffer D 7.4)  centrifuged discarded  (10 mM  Tris,  and mixed f o r 2 h r s .  were removed by c e n t r i f u g a t i o n a t 10,000 x g f o r  50  5 mins. and then the r i b o s o m a l mixture was 105,000 x g f o r 7-10  hours.  The  centrifuged at  supernatant was  and the r i b o s o m a l p e l l e t d i s s o l v e d i n b u f f e r E 10 mM  Mg(OAc) , 0.5-1.0 M NH„C1, 2  pH 7.4)  discarded  (10 mM  Tris,  and mixed f o r 2 h r s .  Aggregates  were removed by c e n t r i f u g a t i o n a t 10,000 x g f o r  5 mins and  then the r i b o s o m a l mixture was  105,000 x g f o r 5 h r s .  The  centrifuged at  s u p e r n a t a n t was  d i s c a r d e d and  mixing of the p e l l e t w i t h b u f f e r E and subsequent t i o n was  repeated t h r e e times more.  finally  dissolved i n buffer F  Mg(OAc) , pH 7.6) 2  (10 mM  The  and mixed f o r 2 h r s .  (the  (WRib) was  checked  The  was  NtUCl, 10  Aggregates  by c e n t r i f u g a t i o n a t 10,000 x g f o r 5 mins. mixture  centrifuga-  ribosomal p e l l e t  T r i s , 10 mM  the  mM  were removed  ribosomal  f o r amino a c i d a c c e p t o r a c t i v i t y  method of which w i l l be o u t l i n e d i n d e t a i l below) and  finally  s t o r e d a t -70°  i n s m a l l 4-ml  v i a l s c o n t a i n g 1 ml  aliquots. Assay  f o r Ribosomal Ribosomal  of  1  Activity  a c t i v i t y was  and Amino A c i d I n c o r p o r a t i o n determined  by f o l l o w i n g the  uptake  "*C-Phe i n a p o l y U-dependent p h e n y l a l a n i n e i n c o r p o r a t i o n  system.  The  assay system  ( t o t a l volume 250  yl) :  c o n t a i n e d the f o l l o w i n g  50 mM 14 mM 100 mM 6 mM 2 mM 100 nM 5 y l ^C-Phe 50 yg 50 y l 100 y l 10 y l  T r i s , pH 7.6 Mg(OAc) NH„C1 ATP GTP u n l a b e l l e d Phe (1:2 w i t h H 0) poly U WRib S100 S100A 2  2  components  51 The p r e v i o u s l y  f r o z e n components were thawed and then  p l a c e d on i c e and a l l a d d i t i o n s were made a t 0 ° - 4 ° . and S100A v i a l s were d i s c a r d e d a f t e r each usage. a d d i t i o n s were completed  The S100  After a l l  (the C-Phe was added l a s t ) t h e ll,  1  tubes were i n c u b a t e d a t 37° f o r 15 mins.  A f t e r the incubation  the tubes were q u i c k l y p l a c e d i n i c e and mixed w i t h 3 ml o f i c e c o l d TCA ( t r i c h l o r o a c e t i c a c i d ) . h e a t e d a t >90° f o r 20 mins. a t 0° f o r 15 mins.  The tubes were then  They were then removed and k e p t  The p r o t e i n was then c o l l e c t e d on M i l l i -  pore f i l t e r s which were subsequently d r i e d under a h e a t i n g lamp.  The d r i e d f i l t e r s were p l a c e d i n s c i n t i l l a t i o n  and 5 ml of s c i n t i l l a t i o n f l u i d  vials  [ c o n t a i n i n g 3g o f PPO(2,5-  d i p h e n y l o x a z o l e ) and 0.1 g of dimethyl-POPOP(l,4-bis-2-(4methyl-5-phenyloxazoly1)-benzene  p e r l i t e r o f t o l u e n e ] was  added and counted i n a s c i n t i l l a t i o n c o u n t e r . Assay  f o r Amino A c i d A c c e p t o r A c t i v i t y The assay system was e s s e n t i a l l y t h a t o f K e l l e r  was  as f o l l o w s  :  (229)  and  0.2 ml o f assay mix (250 mM T r i s , 100 mM M g , 12.5 mM ATP, pH 7.6) 0.7 ml tRNA 0.1 ml enzyme ( f r e s h l y prepared) 5 u l ^C-amino a c i d mixture (1:2 w i t h H 0) ++  2  A l l additions  2  were made a t 0 ° - 4 ° .  i n c u b a t e d a t 37° f o r 20 mins.  The tubes were  A f t e r the i n c u b a t i o n t h e tubes  were q u i c k l y p l a c e d i n i c e and mixed w i t h 2 mis of i c e c o l d TCA  t o s t o p the r e a c t i o n .  The c o n t e n t s were then c o l l e c t e d on  M i l l i p o r e f i l t e r s which were p l a c e d i n s c i n t i l l a t i o n v i a l s and d r i e d w i t h the a i d o f a heat lamp. 1  '  2  See Appendix,  page 163  F i v e mis. o f t o l u e n e  52  scintillation  f l u i d was added and the v i a l s were then  on a s c i n t i l l a t i o n  counted  counter.  P r e p a r a t i o n o f E . c o l i Aminoacyl-tRNA  synthetases  E. c o l i aminoacyl-tRNA s y n t h e t a s e s were prepared by a two-step  procedure.  Step  (a) was employed to remove a l l the  tRNA w h i l e s t e p (b) was used t o remove a l l low m o l e c u l a r weight m a t e r i a l . of  I n t h i s way a r e l a t i v e l y c l e a n p r e p a r a t i o n  s y n t h e t a s e s c o u l d be i s o l a t e d ,  (a) Treatment  on D E A E - c e l l u l o s e column  A f i b e r o u s form o f the r e s i n , DE-22 was used.  The DE-22  was washed w i t h IN NaOH and then w i t h IN HC1 by the standard procedures  as d e s c r i b e d i n the Whatman i n f o r m a t i o n l e a f l e t .  DE-22, i n the f u l l y p r o t o n a t e d form, was e q u i l i b r a t e d a t the pH meter by s t i r r i n g w i t h enough NaOH so t h a t the pH on continued s t i r r i n g  remained  a t about 7.  T h i s produces a  m a t e r i a l w i t h a h i g h c a p a c i t y t o b i n d tRNA (mean s m a l l i o n c a p a c i t y 1.0 meq/g d r y r e s i n ) . column volume o f 0.15 ml.  Each mg o f tRNA r e q u i r e s a  The column was used o n l y once and  then the r e s i n was d i s c a r d e d . The  f o l l o w i n g steps were a l l c a r r i e d o u t a t 0°-4°.  Three  mis o f S100 (approximately 0.6 mg RNA/ml) was thawed o u t and 0.75 ml o f 1.0 M KC1 was added t o g i v e 3.75 ml o f 0.2 M K C l . T h i s enzyme mixture was then p u t through  a 0.5 ml DE-22 column  p r e v i o u s l y e q u i l i b r a t e d w i t h 10 ml b u f f e r G (0.2 M K C l , 10 mM Tris,  pH 7.4) .  The f i r s t  0.4 ml o f e f f l u e n t  (the v o i d volume)  was d i s c a r d e d and then the next 3.7 mis was c o l l e c t e d and put  53 through a Sephadex G-25 column (see below). (b) Treatment  on Sephadex G-25 Column  The Sephadex G-25 was prepared as d e s c r i b e d i n the Pharmacia handbook, "Sephadex-gel f i l t r a t i o n i n theory and practice."  The f o l l o w i n g steps were c a r r i e d o u t a t 0°-4°.  A  20 ml Sephadex G-25 column was prepared and e q u i l i b r a t e d w i t h 35 ml o f f r e s h l y prepared b u f f e r H (20 mM T r i s , 10 mM  cysteine,  pH 7.4). Three mis o f e f f l u e n t from the column w i t h b u f f e r H.  through  C o l l e c t i o n o f e f f l u e n t was s t a r t e d  as soon as the e f f l u e n t from o f the column.  (a) above was e l u t e d  (a) was p i p e t t e d onto  the top  P r o t e i n s t a r t e d t o emerge from the column a t  the v o i d volume which was about 35% o f the column volume. S i n c e the v o i d volume v a r i e d w i t h the packing o f the column, s m a l l a l i q u o t s were removed from the column and t e s t e d w i t h IM HClOi,.  A d i s t i n c t t u r b i d i t y o c c u r r e d when the c o n c e n t r a t i o n  o f p r o t e i n i n the t e s t s o l u t i o n was 0.2 mg/ml o r more.  At  t h i s p o i n t a f u r t h e r 4.2 ml o f e f f l u e n t was c o l l e c t e d (or u n t i l IM HC1CU t e s t i s negative) and made 40% w i t h r e s p e c t to g l y c e r o l and s t o r e d a t - 2 0 ° .  I t has been found t h a t  aminoacyl-tRNA s y n t h e t a s e s maintained s t o r e d f o r one year i n g l y c e r o l . s y n t h e t a s e the a c t i v i t y  certain  t h e i r a c t i v i t y when  I n the case o f seryl-tRNA  i n c r e a s e d i n the presence o f g l y c e r o l  w h i l e w i t h some o t h e r s the a c t i v i t y decreased i n absence o f glycerol  (279) .  The enzyme mixture was used as prepared  w i t h o u t p r i o r removal  o f the g l y c e r o l .  The column was washed  54 w i t h 30 ml o f d i s t i l l e d water to wash out the b u f f e r and ready f o r use  was  again.  Preparation of Benzoylated DEAE-cellulose (BD-cellulose) BD-cellulose  ( f u l l y b e n z o y l a t e d ) , prepared by  reaction  of D E A E - c e l l u l o s e w i t h b e n z o y l c h l o r i d e as d e s c r i b e d by G i l l a m e t a l . (228) was a 50 mesh (0.3 mm  ground and s i e v e d i n the wet  opening) s c r e e n and f r e e d o f f i n e  by repeated s e t t l i n g and d e c a n t a t i o n . the removal  state  (0.1-0.5 M)  e x c e s s i v e g e n e r a t i o n of f i n e s .  particles  During the s i e v i n g  of the f i n e s , the B D - c e l l u l o s e was  solutions containing dilute  through  maintained i n  NaCl to p r e v e n t  The B D - c e l l u l o s e was  packed  i n t o columns by adding a s l u r r y o f the r e s i n i n 2 M  NaCl  (which had been f r e e d of trapped a i r by evacuation)  to a  column h a l f f i l l e d w i t h 2 M NaCl. s e t t l e u n t i l approximately The column stop-cock was  2-3  s l u r r y was  allowed to  cm o f B D - c e l l u l o s e had  then opened b u t the l i q u i d  was  always kept above the packed  The  s l u r r y was  obtained.  The  s u r f a c e o f the  column was  level  was  washed w i t h 2 M NaCl  the e l u a t e had a c c e p t a b l y low absorbance  (A 6o 2  F o l l o w i n g these washing s t e p s , the column was  packed.  exchanger.  added u n t i l the d e s i r e d depth o f bed  The packed  and  nm  until  0.025).  equilibrated  w i t h the s o l u t i o n used t o s t a r t the e l u t i o n and the tRNA ( d i s s o l v e d i n the l a t t e r s o l u t i o n or a s o l u t i o n of e q u a l o r l e s s c o n d u c t i v i t y ) was  a p p l i e d to the column.  After a brief  r i n s i n g w i t h the s o l u t i o n used to e q u i l i b r a t e the column, p o s i t i v e l i n e a r g r a d i e n t s o f NaCl were a p p l i e d i n the u s u a l  55  manner (292,  293) .  Treatment of Ribosomes w i t h The 5 ml) (PM) A.  f o l l o w i n g i n c u b a t i o n mixtures  were prepared  and made 0.5  mM mM mM mM mM yl yl mg mM  B. 10 Tris 6 ATP 14 Mg(OAc) 100 NIUC1 GTP 2 S100 100 S100A 20 2 WRib amino a c i d mix mixture A  and p r e i n c u b a t e d  (148,  T r i s , 0.1  p e l l e t was mM  p e l l e t was  2  mixed i n 10 mM  then i n c u b a t e d  f o r 15 mins.  was  a t 30°  7.6  Tris Mg(OAc) NHUC1 GTP S100A WRib  brought up to pH  7.6  a d d i t i o n and  Following  and d i a l y z e d o v e r n i g h t  Mg(OAc)  (148,  b u f f e r e d a t pH  2  272)  was  the  mM  against  7.8  the (buffer  brought up to pH  f o r 15 mins w h i l e  brought up  Following  The  2  mM mM mM mM yl mg  c e n t r i f u g e d a t 105,000 x g f o r 5  to pH  7.6  and  7.6  i n c u b a t i o n mixture  incubated  at  30°  the i n c u b a t i o n both mixtures were made  molar with r e s p e c t to NHi,Cl and  g for 5 hrs.  was  5 14 100 2 100 13  a g a i n s t the same b u f f e r .  I n c u b a t i o n mixture B  272)  272)  C.  A f t e r c e n t r i f u g a t i o n a t 105,000 x g,  A)and d i a l y z e d o v e r n i g h t  (148,  Tris ATP Mg(OAc) NH^Cl GTP S100A WRib S100  then mixed i n b u f f e r I c o n t a i n i n g 10  Mg(OAc) , pH  the same b u f f e r .  one  r e s p e c t to puromycin  f o r 30 mins a t 37°.  i n c u b a t i o n the mixture was  C  with  f o r 5 mins a t 37° p r i o r to PM  then f u r t h e r i n c u b a t e d  The  mM mM mM mM mM yl mg ml  2  Incubation  and  mM  ( i n a t o t a l volume of  :  10 6 14 100 2 2 100 46 200  hrs.  Puromycin  c e n t r i f u g e d a t 105,000 x  p e l l e t s were mixed i n 10 mM  Mg(OAc)  2  2  56  buffered  a t pH 7.6 ( b u f f e r F) and s t o r e d a t - 2 0 ° .  Treatment o f Ribosomes w i t h P e r i o d a t e The  WRib were i n c u b a t e d  alkaline buffer  f o r two hours a t 37° i n a m i l d  (0.5 M T r i s . pH 9.0).  the bound tRNA o f amino a c i d s  The r i b o s o m a l  This process s t r i p p e d  (273, 294). The l i b e r a t e d  amino a c i d s were removed by d i a l y s i s at 4°.  (HIOi»)  a g a i n s t d i s t i l l e d water  p e l l e t was o b t a i n e d  by c e n t r i f u g a t i o n  a t 105,000 x g and mixed i n a b u f f e r c o n t a i n g pH  5.0 p l u s 1.1 umoles NalOif/mg RNA.  incubated  100 mM KOAc,  T h i s m i x t u r e was  a t room temperature f o r 45 minutes i n the d a r k .  A f t e r the i n c u b a t i o n p e r i o d 1.0 ml o f g l y c e r o l was added t o reduce any u n r e a c t e d p e r i o d a t e f o r an a d d i t i o n a l 30 mins.  and the mixture was  reincubated  The m i x t u r e was spun a t 105,000  x g and the p e l l e t mixed i n 10 mM Mg(OAc)2 b u f f e r e d  a t pH 7.6  ( b u f f e r F) . A n a l y t i c a l Polyacrylamide  Gel Electrophoresis  (a) A m o d i f i c a t i o n o f the D a v i s system was used f o l l o w i n g stock bottles  (221).  The  s o l u t i o n s were p r e p a r e d and s t o r e d i n brown  i n the c o l d room. Stock (A) IN HC1 48 ml Tris 36.6 gm TEMED 0.23 gm 8M u r e a t o 100 ml (pH 8.9)  Solutions  (B) IN HC1 48 ml Tris 5.98 gm TEMED 0.46 gm 8M u r e a t o 100 ml (pH 6.7)  57  Stock S o l u t i o n s continued (D)  (C) acrylamide 28.0 gm Bis 0.735 gm 8M u r e a t o 100 ml  acrylamide 10.0 gm Bis 2.5 gm 8M urea t o 100 ml  (E)  (F)  riboflavin 4 mg 8M u r e a t o 100 ml The of  f o l l o w i n g working  sucrose 40 gm 8M urea t o 100 ml  s o l u t i o n s were p r e p a r e d t h e day  the r u n and then d i s c a r d e d . Small-pore s o l u t i o n #2  Small-pore s o l u t i o n #1 1 part A 2 parts C 1 p a r t 7M u r e a pH 8.9  Large-pore solution  ammonium p e r s u l f a t e 0.14 gm 7M urea t o 100 ml  Stock buffer solution for reservoirs*  2 parts B T r i s 6.0 gm 4 p a r t s D g l y c i n e 28.8 gm 2 parts E water t o 1 l i t e r 2 parts F pH 8.3 2 p a r t s sample  * d i l u t e d 1:10 w i t h water The g e l s were p r e p a r e d i n c y l i n d r i c a l g l a s s tubes, 0.5 x 10 cms.  The c l e a n g l a s s tubes were f i r s t p l a c e d i n an u p r i g h t  p o s i t i o n i n a tube s t a n d . cementing  Stands a r e c o n v e n i e n t l y made by  hollow rubber s t o p p e r s , f o r example, the B-D Vacu-  t a i n e r s t o p p e r s , i n a s i n g l e row, a few cms a p a r t , w i t h the c l o s e d end down, t o a f l a t p i e c e o f p l a s t i c . the cap s h o u l d f i t snugly around of  the i n g r e d i e n t s .  the g e l tube t o p r e v e n t leakage  The l a r g e pore s o l u t i o n was p r e p a r e d and  run i n t o the g l a s s tubes. top  The open end o f  A water l a y e r was then p l a c e d on  o f t h e g e l s o l u t i o n i n such a manner as n o t t o d i s t u r b  the g e l s u r f a c e .  The tube stand was now p l a c e d d i r e c t l y  under  a f l u o r e s c e n t b u l b f o r about 30 mins t o a l l o w f o r p h o t o p o l y -  58  merization.  F o l l o w i n g p h o t o p o l y m e r i z a t i o n the water l a y e r  removed and the g e l tubes c o m p l e t e l y gel  f i l l e d with  s o l u t i o n , prepared by mixing e q u a l volumes of  s o l u t i o n s #1 and #2.  was  small-pore small-pore  The g e l s were allowed to p o l y m e r i z e  w h i l e b e i n g p r o t e c t e d from s t r o n g l i g h t . F o l l o w i n g p o l y m e r i z a t i o n o f the g e l s , the g l a s s tubes were removed from the tube stand and p l a c e d i n the c o l d room on an e l e c t r o p h o r e t i c apparatus,  as i l l u s t r a t e d by Davis  c o n t a i n i n g the a p p r o p r i a t e b u f f e r . a t t a c h e d to the apparatus  The e l e c t r o d e s were  and to a power supply s e t a t a  c o n s t a n t c u r r e n t o f 5 mA/tube.  P r i o r to t u r n i n g on the power  s u p p l y , a drop o r two o f 0.001% bromophenol b l u e was s e r v e as the marker  (221),  added to  dye.  A f t e r the run the g l a s s tubes were removed from e l e c t r o p h o r e t i c apparatus  and  the g e l s were  the  subsequently  removed by rimming the tubes under water w i t h a w i r e .  The  g e l s were s t a i n e d f o r 1 h r i n a 15% IlAc s o l u t i o n c o n t a i n i n g 2% a c r i d i n e orange  ( s p e c i f i c f o r n u c l e i c a c i d s ) and  lanthanum a c e t a t e ( f i x a t i v e ) o v e r n i g h t i n 15% HAc  and  (222).  1%  The g e l s were d e s t a i n e d  then s t o r e d i n 7% HAc  i n the c o l d  room. (b) A m o d i f i c a t i o n o f the Dingman and Peacock system used  (223-225).  The  f o l l o w i n g working s o l u t i o n f o r the  p r e p a r a t i o n o f a 10% p o l y a c r y l a m i d e g e l was of  was  the run and then d i s c a r d e d :  prepared  the  day  59  7.9 ml a c r y l a m i d e mixture (19.5% a c r y l a m i d e + 0.5% Bis) 4.65 ml H 0 0.95 ml DMAPN (6.4%) 1.5 ml b u f f e r (undiluted) ( T r i s 108 gm, EDTA diNa 9.3 gm, B o r i c A c i d 55 gm : t o 1 l i t e r w i t h H 0, pH 8.3) 10 mg ammonium p e r s u l f a t e 2  2  The  g e l s were prepared i n c y l i n d r i c a l g l a s s tubes, 0.5  x 10 cms.  The g l a s s tubes were f i r s t p l a c e d  p o s i t i o n i n a tube s t a n d .  i n an u p r i g h t  The g e l m i x t u r e was p r e p a r e d ,  mixed and r u n i n t o the g l a s s tubes f o r a d i s t a n c e o f 7 cms. A water l a y e r was then p l a c e d on top o f the g e l s o l u t i o n i n such a manner as n o t t o d i s t u r b the g e l s u r f a c e were a l l o w e d t o p o l y m e r i z e .  and the g e l s  A f t e r polymerization,  l a y e r was removed, and the g l a s s tubes were f i l l e d diluted buffer  (1:10 w i t h H 0 ) .  from the tube stand  2  and p l a c e d  with  The tubes were then removed i n the c o l d room on an  e l e c t r o p h o r e t i c a p p a r a t u s , as d e s c r i b e d the same d i l u t e d b u f f e r .  the water  i n (a), containing  The tubes were then p r e e l e c t r o -  phoresed f o r 30 mins a t 5mA/tube. After preelectrophoresis,  the b u f f e r was removed from  the g l a s s tubes and up t o 200 y l o f sample i n 10% in  sucrose,  the presence o f a drop o r two o f bromophenol b l u e ,  l a y e r e d onto the g e l s u r f a c e .  was  The r e s t o f the g l a s s tube was  f i l l e d w i t h d i l u t e d b u f f e r so as n o t t o d i s t u r b the sample l a y e r and then p l a c e d apparatus.  i n the c o l d room on the e l e c t r o p h o r e t i c  The e l e c t r o d e s were attached  and e l e c t r o p h o r e s i s  was c a r r i e d o u t a t 10 v o l t s p e r cm l e n g t h o f g l a s s A f t e r the r u n the g e l s were o b t a i n e d  tube.  as d e s c r i b e d  i n (a)  60  and  then s t a i n e d f o r 1 h r i n 0.2%  an a c e t a t e b u f f e r were d e s t a i n e d  (0.4  methylene b l u e d i s s o l v e d i n  M NaOAc, 0.4  overnight  M HAc,  pH  4.7).  i n d i s t i l l e d water and  The  gels  then s t o r e d  i n d i s t i l l e d water i n the c o l d room. T h i s was  found to be  o f low m o l e c u l a r weight RNA  the b e s t method f o r the and was  separation  subsequently used f o r the  i d e n t i f i c a t i o n o f these RNAs i n the R e s u l t s  section.  (c) A m o d i f i c a t i o n o f the method o f Moriyama e t a l . (195) used.  The  o n l y d i f f e r e n c e between t h i s procedure and  used i n (b) was Preparation The 10%  the type o f g e l  o f the  then  that  preparation.  gel  f o l l o w i n g working s o l u t i o n f o r the p r e p a r a t i o n  polyacrylamide  was  g e l was  p r e p a r e d the day  of the run  of a and  discarded. 0.5 ml o f 10% ammonium p e r s u l f a t e and 10 y l TEMED was added to 25 ml o f 10% g e l s o l u t i o n (9.75% a c r y l a m i d e + 0.25% B i s i n 40 mM T r i s , 20 mM NaOAc, 2 mM EDTA, pH 7.4, 25°) p r e v i o u s l y degassed f o r a few seconds.  Preparative  Polyacrylamide Gel  A Canalco p r e p a r a t i v e  Electrophoresis  disc electrophoresis  w i t h a PD-2/320 upper column was stock  s o l u t i o n s were prepared and  used  (194).  apparatus  The  following  s t o r e d i n brown b o t t l e s i n  the c o l d room. (A) IN HC1 48 ml Tris 36.6 g TEMED 0.23 g 8M urea to 100 ml (pH 8.9)  (C) a c r y l a m i d e 28.0 g Bis 0.735 g 8M urea to 100 ml  The of  f o l l o w i n g working  s o l u t i o n s were prepared the day  the r u n and then d i s c a r d e d .  Small-pore solution #1  Small-pore solution #2 ammonium persulfate 0.14 g 7M u r e a t o 100 ml  1 part A 2 parts C 1 p a r t 7M urea pH 8.9  • d i l u t e d 1:10 w i t h In  Elution buffers  (1) 1 p a r t A 7 parts H 0 (2) 40 mM T r i s 20 mM NaOAc 2 mM EDTA, pH 7.2 2  Stock b u f f e r s o l u tions f o r electrode compartments* (1) T r i s 6.0 g g l y c i n e 28.8 g H 0 to 1 l i t e r , pH 8.3 (2)upper e l e c t r o d e buffer : 40 mM T r i s 20 mM NaOAc 2 mM EDTA, pH 7.2 lower e l e c t r o d e buffer : 80 mM T r i s 40 mM NaOAc 4 mM EDTA, pH 7.2 2  H0 2  o r d e r t o prepare the g e l , the bottom end o f the upper  column was capped w i t h a square o f Saran Wrap.  The column  was clamped i n a v e r t i c a l p o s i t i o n w i t h the bottom end r e s t i n g on a f l a t to  surface.  The capped upper column was f i r s t  filled  the mark w i t h s m a l l - p o r e g e l s o l u t i o n , prepared by m i x i n g  e q u a l volumes o f s m a l l pore s o l u t i o n s #1 and #2 and then a w a t e r - l a y e r was p l a c e d on top o f the g e l s o l u t i o n i n such a manner as n o t t o d i s t u r b the g e l s u r f a c e .  The column was then  p l a c e d between two f l u o r e s c e n t b u l b s . F o l l o w i n g p o l y m e r i z a t i o n the water l a y e r was removed a l o n g w i t h the Saran Wrap and the upper column was f i l l e d the assembly.  The sample i n 10% sucrose was c a r e f u l l y  into  layered  62 on top of the g e l s u r f a c e .  The upper e l e c t r o d e b u f f e r was  then s l o w l y added, so as not t o d i s t u r b the g e l s u r f a c e , u n t i l the upper column r e s e r v o i r was r e s e r v o i r was  filled.  The  lower e l e c t r o d e  a l s o f i l l e d w i t h the a p p r o p r i a t e b u f f e r and  then b o t h e l e c t r o d e s were connected a c o n s t a n t c u r r e n t o f 7mA.  The  t o a power s u p p l y s e t a t  a p p r o p r i a t e e l u t i o n b u f f e r was  pumped i n and o u t a t a flow r a t e o f 60 ml per h r , and by means o f a f r a c t i o n c o l l e c t o r . by means of two  The  system was  s e p a r a t e c o o l i n g systems.  collected  kept a t 4°  A marker dye  was  not used i n o r d e r to a v o i d the p o s s i b l e e f f e c t s o f i t s c o n t r i b u t i o n t o the A 6 o  r e a d i n g s o f the c o l l e c t e d e f f l u e n t .  2  run was  t e r m i n a t e d when the A 6 o 2  r e a d i n g s o f the  The  effluent  had become n e g l i g i b l e . P r e p a r a t i o n o f 30S  and 50S  s u b u n i t s from Ribosomes  The p r e p a r a t i o n o f r i b o s o m a l s u b u n i t s was t h a t o f Cannon e t a l . (226). with b u f f e r K  (5 mM  T r i s , pH  The r i b o s o m a l p e l l e t was 7.3  + 0.1  mM  Mg(OAc) ) 2  d i a l y z e d a g a i n s t the same b u f f e r f o r 28 h r s . mixture was  essentially  The  at  30%  sucrose prepared  25,000 rpm  ribosomal  c o l l e c t e d through  20,  25  i n the same b u f f e r and c e n t r i f u g e d  f o r 12 h r s a t 4° i n a Model L  u s i n g a SW-39 r o t o r .  system.  and  then p u t on a 10-30% d i s c o n t i n u o u s sucrose  g r a d i e n t which c o n t a i n e d e q u a l a l i q u o t s o f 10, 15, and  mixed  ultracentrifuge  A f t e r c e n t r i f u g a t i o n , f r a c t i o n s were  the use of the Beckman f r a c t i o n r e c o v e r y  I n t h i s procedure  each tube was  h o l d e r and a r e c o v e r y cap was  p l a c e d i n a tube  screwed on top o f the h o l d e r to  63  m a i n t a i n an a i r - f r e e system.  The tube was p i e r c e d from the  bottom by a n e e d l e , dense s u c r o s e was pumped i n s l o w l y through the needle so as n o t t o d i s t u r b the g r a d i e n t and f r a c t i o n s were c o l l e c t e d  from a rubber tube l e a d i n g from the r e c o v e r y  cap. Preparation o f CsCl Gradient The method was e s s e n t i a l l y t h a t o f Meselson e t a l . and Traub and Nomura (6 0 ) . was  (227)  Each o f the r i b o s o m a l s u b u n i t s  c e n t r i f u g e d through a C s C l g r a d i e n t .  The r i b o s o m a l  s u b u n i t s were d i s s o l v e d i n a b u f f e r made up o f 20 mM T r i s , 40 mM M g C l , pH 7.6. One ml o f each s u b u n i t s u s p e n s i o n was 2  mixed w i t h 4.3 ml o f 61% (w/v) C s C l d i s s o l v e d i n the same buffer.  The r e s u l t i n g mixture had an index o f r e f r a c t i o n  r\ * = 1.39 50. 2  A p p r o x i m a t e l y 5.0 ml o f t h i s mixture was p l a c e d  i n a L u s t e r o i d c e n t r i f u g e tube i n a S p i n c o SW-39 r o t o r .  To  i n s u r e the s t a b i l i t y o f the s u b u n i t s , i t was found n e c e s s a r y t o wash the L u s t e r o i d t u b e s .  T h i s was done f o r 1 h r i n  b o i l i n g 1 mM EDTA and then i n b o i l i n g water. was  Centrifugation  c a r r i e d o u t a t 36,000 rpm f o r 36 h r s a t 4 ° .  After  slow  d e c e l e r a t i o n , each L u s t e r o i d tube was withdrawn and p l a c e d i n a tube h o l d e r (Beckman).  The tubes were p i e r c e d from the  bottom w i t h a needle and f r a c t i o n s were c o l l e c t e d by g r a v i t y .  I s o l a t i o n o f T o t a l RNA from Ribosomes Ribosomes were mixed w i t h b u f f e r L (10 mM T r i s , pH 7.6, 10 mM M g  + +  + 0.5% sodium d o d e c y l s u l f a t e  (SDS)) and an e q u a l  64 volume o f w a t e r - s a t u r a t e d phenol f o r 20 mins a t room temperature. fugation  The aqueous l a y e r was c o l l e c t e d a f t e r c e n t r i -  a t 5,000 x g f o r 10 mins.  w i t h h a l f a volume o f b u f f e r was  The phenol l a y e r was mixed  f o r 20 mins.  r e c o v e r e d a f t e r c e n t r i f u g a t i o n and p o o l e d w i t h the  initial  aqueous l a y e r .  I t was then mixed w i t h an e q u a l volume  o f phenol f o r 20 mins a t room temperature. was  The aqueous l a y e r  The aqueous l a y e r  r e c o v e r e d a f t e r c e n t r i f u g a t i o n and the phenol removed by  ether e x t r a c t i o n .  R e s i d u a l e t h e r was removed by b u b b l i n g N  through the s o l u t i o n . added t o g i v e  Sodium a c e t a t e  2  (1.5 M, pH 5.2) was  a 2% s o l u t i o n and the RNA p r e c i p i t a t e d by  a d d i t i o n o f 3 volumes o f c o l d e t h a n o l .  The t o t a l RNA was  c o l l e c t e d by c e n t r i f u g a t i o n and d i s s o l v e d i n e i t h e r 10 mM Mg(OAc)  2  buffered  Preparation The above.  a t pH 7.6 ( b u f f e r F) o r w a t e r .  o f High and Low M o l e c u l a r Weight RNA from Ribosomes  t o t a l RNA from ribosomes was p r e p a r e d as d e s c r i b e d The RNA was d i s s o l v e d  i n 0.1 M T r i s , pH 7.5 and then  t h i s m i x t u r e was made 2 M w i t h r e s p e c t 4°  f o r 2 days.  t o NaCl and k e p t a t  The p r e c i p i t a t e c o n t a i n i n g  the h i g h m o l e c u l a r  weight RNA was s e p a r a t e d from the s u p e r n a t a n t c o n t a i n i n g low m o l e c u l a r weight RNA by low speed c e n t r i f u g a t i o n . to ensure a b e t t e r s e p a r a t i o n weight m a t e r i a l ,  In order  o f h i g h and low m o l e c u l a r  the RNA p r e c i p i t a t e was d i s s o l v e d  i n the  above b u f f e r  and r e p r e c i p i t a t e d when the b u f f e r was made 2 M  with respect  to NaCl.  The p r e c i p i t a t e , c o n t a i n i n g  m o l e c u l a r weight RNA, was d i s s o l v e d  high  i n 50 mM NaCl and s t o r e d  65  at -20°. Mg(OAc)  The s u p e r n a t a n t was made 10 mM w i t h r e s p e c t t o and then NaOAc (1.5 M, pH 5.2) was added t o g i v e a 2%  2  solution.  The RNA was subsequently p r e c i p i t a t e d w i t h 3 volumes  o f c o l d ETOH.  The low m o l e c u l a r weight RNA was c o l l e c t e d by  centrifugation  and d i s s o l v e d  further  characterized  electrophoresis.  i n 50 ml NaCl.  I t was subsequently  on Sephadex G-100 and by p r e p a r a t i v e g e l  66  66  CHART I I * Procedures f o r p r e p a r i n g E. c o l i E.  ribosomes  coli a) ground w i t h g l a s s beads i n b u f f e r A (10 mM Mg(OAc) ) b) c e n t r i f u g e d a t 10,000 x g 2  cellular  debris  supernatant a) i n c u b a t e d w i t h DNase b) c e n t r i f u g e d 2x a t 30 ,000 x g  supernatant - i a) mix w i t h b u f f e r H a) mixed w i t h b u f f e r M (5mM Mg(OAc) / 250mM NH^Cl 2  ribosomal p e l l e t _ a) mixed w i t i b u f f e r G b) c e n t r i f u g e d a t 105,000 x g  b) Put on D E A E - c e l l u l o s e column supernatant e q u i l i b r a t e with b u f f e r M and wash w i t h same b u f f e r  b) c e n t r i f u g e d a t 105,000 x g  1  precipitate  supernatant centrifuged at 105 ,000 x g •ribosomal p e l l e t supernatant a) mixed w i t h (S100) b u f f e r B (0.1 mM Mg(OAc) ) b) c e n t r i f u g e d a t 105,000 x g  c) steps a) and b) repeated 2x  ribosomal p e l l e t (RSII)  2  ribosomal p e l l e t S100A a) mixed w i t h b u f f e r C (1 mM Mg(OAc) ) b) c e n t r i f u g e d a t 105,000 x g  c) e l u t e w i t h b u f f e r N (lOmM Mg(OAc) , 1.0M NH^Cl  2  2  ribosomal p e l l e t supernatant a) mixed w i t h b u f f e r D (5 mM Mg(OAc) ) b) c e n t r i f u g e d a t 105,000 x g i ribosomal p e l l e t supernatant a) mixed w i t h b u f f e r E (10 mM Mg(OAc) ) b) c e n t r i f u g e d a t 105,000 x g c) r e p e a t e d a) and b) 3x 2  ribosomal suspension | c e n t r i f u g e d a t 105,000 x g P-i  ribosomal p e l l e t mixed w i t h buffer F  2  ^  supernatant ]  1  ribosomal p e l l e t supernatant 1 lmixed w i t h b u f f e r F (10 mM Mg(OAc) )  I ribosomal  2  suspension (WRib) a) Put on D E A E - c e l l u l o s e column e q u i l i b r a t e d w i t h b u f f e r M and wash w i t h same b u f f e r b) e l u t e d w i t h b u f f e r N  r i b o s o m a l suspension (RSIII)  r i b o s o m a l suspension | c e n t r i f u g e d a t 105,000 x g j  pi  ribosomal p e l l e t 1 mixed with b u f f e r F  I  supernatant  r i b o s o m a l suspension (RSI) * B u f f e r A-H, M-N I  I i  described  previously i n L i s t of Buffers.  67  TABLE I The a c t i v i t y o f ribosomes prepared by different  Ribosome p r e p a r a t i o n * Experiment I  WRib RSI WRib RSI  Experiment I I  Concentration A 2 6 0 units  Activity**  50 52 20 18  H-WRib*** RSII RSIII WRib  3  methods  29 74 16 33  11.6 10.9 9.6 10.0  * The d e s c r i p t i o n o f the p r e p a r a t i o n s  23 20 24 2  are given i n Chart I I .  ** A c t i v i t y determined by f o l l o w i n g the i n c o r p o r a t i o n o f **C1  Phe i n a p o l y U - d i r e c t e d s y n t h e s i s o f described  i n M a t e r i a l s and Methods.  polyphenylalanine  The a c t i v i t y o f t h e  b l a n k c o n t a i n i n g no ribosomes was s e t a t one (200 cpm). ***  This preparation  i s i d e n t i c a l t o WRib except the c e l l s  o r i g i n a t e d from a p y r i m i d i n e - r e q u i r i n g mutant o f E_^ c o l i B (ATCC 13135) grown i n the presence o f H - u r a c i l . 3  68  RESULTS The A c t i v i t y The  of Ribosome P r e p a r a t i o n s  ribosomes  prepared as o u t l i n e d  i n Materials  and  Methods were found t o have an amino a c i d a c c e p t o r a c t i v i t y 50-100 f o l d over the b l a n k l e v e l which was A260/A280  was  r a t i o of these ribosomes was  s e t a t one.  The  found t o be 2:1  and  comparable and i n many cases g r e a t e r t o o t h e r r e p o r t e d  preparations  (282) .  These ribosomes  used i n a l l f u t u r e experiments. t h r e e o t h e r procedures  Ribosomes were p r e p a r e d by  as o u t l i n e d i n C h a r t I I and compared  w i t h WRib w i t h r e s p e c t t o a c t i v i t y m o l e c u l a r weight RNA The RSI column was still  (WRib) were s u b s e q u e n t l y  (Table I) and bound  low  (Figure 1).  (Table I) which had been put through a DE-22  much more a c t i v e than WRib.  l e s s than t h a t of the o r i g i n a l  This a c t i v i t y  was  fresh preparation  prepared two months e a r l i e r which showed 8 2 - f o l d amino a c i d acceptor a c t i v i t y  over the b l a n k l e v e l .  T a b l e I , the a c t i v i t y of the ribosomes c o n c e n t r a t i o n of the ribosomes  In experiment  I,  i n c r e a s e d as the  (measured i n  A 6o 2  units)  increased. In experiment  I I , Table I, f r e s h l y prepared  WRib from a p y r i m i d i n e - r e q u i r i n g fold  labelled  c o l i mutant had o n l y a 23-  i n c r e a s e i n a c t i v i t y over the b l a n k .  Perhaps  this  was  due t o the type of c e l l used, the s t a g e of growth a t which the c e l l s were h a r v e s t e d or the manner i n which the were prepared  (see M a t e r i a l s and Methods).  A t the p a r t i c u l a r  c o n c e n t r a t i o n used, the H-WRib, RSII and RSIII had 3  ribosomes  virtually  FIGURE 1 Photograph 7 cm)  o f p o l y a c r y l a m i d e g e l (10% g e l , pH  8.3,  and 0.5  e l e c t r o p h o r e s i s p a t t e r n s o f low m o l e c u l a r weight  o b t a i n e d from d i f f e r e n t p r e p a r a t i o n s o f E ^ c o l i  x  RNA  ribosomes.  From l e f t t o r i g h t *: WRib, RSI, RSII, RSIII and the c o n t r o l (commercial E ^ c o l i  *  tRNA).  The  a b b r e v i a t i o n s are those d e s i g n a t e d i n C h a r t I I .  The  4S and 5S RNA  r e g i o n s i n d i c a t e d have been predetermined  by numerous i n v e s t i g a t o r s and v e r i f i e d by the use o f approp r i a t e standards  (223-225).  To f a c e page 69  69  70  t h e same a c t i v i t y . was  The WRib w h i c h showed o n l y s l i g h t  activity  t h e same as t h a t used i n t h e f i r s t e x p e r i m e n t , T a b l e  except  t h a t i t had been thawed and  Perhaps t h i s was lowered  one  f r o z e n one  I,  more t i m e .  o f t h e reasons f o r i t s d r a s t i c a l l y  activity.  F i g u r e 1 shows the p o l y a c r y l a m i d e  gel electrophoresis  p a t t e r n s o f t h e low m o l e c u l a r w e i g h t RNA  o b t a i n e d from t h e  v a r i o u s ribosome p r e p a r a t i o n s o u t l i n e d i n C h a r t I I . appears t o be much l e s s 4S and  4.5S  w h i c h have been washed through  a DEAE-cellulose  1 M NHi»Cl.  T h i s may  There  RNA* bound t o ribosomes column w i t h  be c o r r e l a t e d w i t h the i n c r e a s e d  r i b o s o m a l a c t i v i t y o b s e r v e d under t h e s e c o n d i t i o n s (Table I ) . The  r e g i o n d e s i g n a t e d X may  be 6S  RNA.  A t t e m p t s t o Remove Bound tRNA from Ribosomes The WRib were t r e a t e d under v a r i o u s c o n d i t i o n s i n an effort  t o remove a l l t h e bound tRNA.  (a) Puromycin  (PM)  The WRib were t r e a t e d w i t h PM  ( r e f e r r e d t o as PM-WRib)  as d e s c r i b e d i n M a t e r i a l s and Methods. p e r i o d , the PM was  A f t e r the i n c u b a t i o n  removed w i t h 1 M NH^Cl.  The  t h e s e PM-WRib t o s u p p o r t p r o t e i n s y n t h e s i s was  c a p a c i t y of found t o be  l e a s t as h i g h as the u n t r e a t e d ones and i n some cases the v i t y was  f o u n d t o be  considerably higher.  at acti-  F i g u r e 2 shows t h e  * This species of RNA (4.5S) has been found to have a mobility greater than 55 RNA but less than 4S RNA (194). Transfer RNA has been found to have the same mobility as 4S RNA and i s considered to be synonymous with 4S RNA. 5S RNA was identif i e d by its mobility r e l a t i v e to 4S RNA in 10% polyacrylamide gels ( 223-225) .  FIGURE 2 Photograph o f p o l y a c r y l a m i d e  g e l (10%, pH 8.3, and 0.5 x  7 cms) e l e c t r o p h o r e s i s p a t t e r n s obtained  from p u r o m y c i n - t r e a t e d  From l e f t  to r i g h t  o f low m o l e c u l a r coli  ribosomes  weight RNA (PM-WRib).  : PM-WRib ( p r e p a r a t i o n B i n M a t e r i a l s and  Methods), PM-WRib ( p r e p a r a t i o n C ) , PM-Mg t r e a t e d WRib ( p r e p a r a t i o n A ) , PM-Mg t r e a t e d WRib ( p r e p a r a t i o n A b u t w i t h twice  t h e PM c o n c e n t r a t i o n )  E. c o l i  and the c o n t r o l  (commercial  tRNA).  To f a c e page 71  71  72 s e p a r a t i o n o f low m o l e c u l a r WRib on p o l y a c r y l a m i d e  w e i g h t RNA from the v a r i o u s PM-  gels.  These PM-WRib were found t o  c o n t a i n bound tRNA. I n some experiments a f t e r t h e i n c u b a t i o n p e r i o d w i t h PM, these PM-WRib were subsequently suspended i n 0.1 mM Mg(OAc)  2  b u f f e r e d a t pH 7.6 ( b u f f e r I ) and d i a l y z e d a g a i n s t t h e same buffer  ( r e f e r r e d t o as PM-Mg t r e a t e d WRib).  t r e a t e d WRib were found t o have very protein synthesis  These PM-Mg  low a b i l i t y  t o support  and t h e r e s u l t s i n F i g u r e 2 showed t h a t  t h e r e was only a t r a c e amount o f tRNA bound t o these PM-Mg t r e a t e d WRib.  T h i s experiment was repeated  concentration,  1 mM and then these PM-WRib were s u b s e q u e n t l y  suspended and d i a l y z e d a g a i n s t b u f f e r I . amount o f bound tRNA was d e t e c t e d WRib ( F i g u r e 2 ) . very (b)  u s i n g a h i g h e r PM  A negligible  i n these PM-Mg t r e a t e d  These ribosomes were a l s o found t o have  low amino a c i d acceptor  activity.*  Periodate WRib were t r e a t e d w i t h  HIOi» i n 100 mM KOAc, pH 5.0 a f t e r  p r i o r m i l d a l k a l i n e h y d r o l y s i s t o s t r i p amino a c i d s bound tRNA as d e s c r i b e d a t e l y HICU completely  i n M a t e r i a l s and Methods.  d i s r u p t e d t h e ribosomal  that the c h a r a c t e r i s t i c ribosomal even a f t e r extended p e r i o d s  from Unfortun-  s t r u c t u r e such  p e l l e t c o u l d not be o b t a i n e d  of u l t r a c e n t r i f u g a t i o n .  There-  f o r e t h i s procedure had t o be abandoned (273) . * P r i o r to the a c t i v i t y studies the PM was r e a d i l y removed from the WRib by incubation in 1 M NH^Cl which breaks the amide linkage between the PM and the WRib,  FIGURE 3 Photograph o f p o l y a c r y l a m i d e g e l (10%, pH 8.3 and 0.5 x 7 cm) e l e c t r o p h o r e s i s p a t t e r n s o f low m o l e c u l a r weight RNA o b t a i n e d from Mg-treated E ^ c o l i  ribosomes.  From l e f t t o r i g h t  t r e a t e d WRib (0.1 mM M g ) , Mg-treated WRib (0.1 mM M g + +  f o l l o w e d by 10 mM M g ) and the c o n t r o l ++  : Mg+ +  (commercial E ^ c o l i  tRNA) .  To f a c e page 73  73  FIGURE 4 The  sedimentation  discontinuous were f i r s t  pattern of  sucrose  gradient.  coli  ribosomes on a 10-30%  The ribosomes  (200A 6o 2  units)  suspended i n b u f f e r K, then d i a l y z e d a g a i n s t the  same b u f f e r and s u b s e q u e n t l y c e n t r i f u g e d through a g r a d i e n t a t 25,000 rpm f o r 12 h r s a t 4 ° . were c o l l e c t e d .  sucrose  One ml f r a c t i o n s  A complete d e s c r i p t i o n i s g i v e n i n M a t e r i a l s  and Methods.  To  f a c e page 74  74  FRACTION NUMBER  75  (c) D i a l y s i s a g a i n s t 0.1 mM Magnesium WRib were mixed i n a b u f f e r c o n t a i n i n g 5 mM T r i s , pH 7.4 p l u s 0.1 mM Mg(OAc) days w i t h was  and d i a l y z e d a g a i n s t the same b u f f e r f o r 2  2  f r e q u e n t changes i n the b u f f e r .  c o l l e c t e d by c e n t r i f u g a t i o n , mixed w i t h  The ribosomal  pellet  10 mM Mg(OAc)  2  b u f f e r e d a t pH 7.6 ( b u f f e r F) and an a l i q u o t was s t o r e d a t -20°.  The r e s t o f the suspension  was d i a l y z e d a g a i n s t the  same b u f f e r o v e r n i g h t and then subsequently E l e c t r o p h o r e s i s i n a 10% p o l y a c r y l a m i d e  frozen a t -20°.  g e l o f the low mole-  c u l a r weight RNA from these M g - t r e a t e d WRib i s shown i n + +  F i g u r e 3.  The WRib d i a l y z e d o n l y a g a i n s t low M g  s t r o n g 5S RNA band b u t very r e l a t i v e t o the 5S RNA. then h i g h M g  + +  + +  showed a  f a i n t 4.5S and 4S RNA bands  The WRib d i a l y z e d a g a i n s t low and  showed a dense 5S RNA band and a s t r o n g 4.5S  RNA band b u t n e g l i g i b l e 4S RNA band r e l a t i v e t o the 5S RNA. I t appears t h a t d i a l y s i s all  the tRNA normally  a g a i n s t low Mg"*""' removes v i r t u a l l y 1  bound t o WRib.  S t u d i e s on the 30S And 50S Ribosomal E. c o l i r i b o s o m a l M a t e r i a l s and Methods.  s u b u n i t s were prepared  as d e s c r i b e d i n  F i g u r e 4 shows the s e p a r a t i o n o f the  s u b u n i t s on a 10-30% d i s c o n t i n u o u s were c o l l e c t e d  Subunits  sucrose  gradient.  from the top as d e s c r i b e d i n the t e x t .  Fractions The 4S  RNA was i d e n t i f i e d as tRNA by the f a c t t h a t i t had amino a c i d acceptor  activity.  F i g u r e 5 shows the r e s u l t s o f e l e c t r o -  p h o r e s i s o f the RNA from the s u b u n i t s gel.  i n a 10% p o l y a c r y l a m i d e  The g e l c o n t a i n i n g only the RNA from the 30S r i b o s o m a l  FIGURE 5 Photograph o f p o l y a c r y l a m i d e  g e l (10%, pH 8.3 and 0.5 x 7 cm)  e l e c t r o p h o r e s i s p a t t e r n s of low m o l e c u l a r from E_;_ c o l i  ribosomal  subunits.  weight RNA o b t a i n e d  From l e f t t o r i g h t  : 30S  RNA*, C s C l - t r e a t e d 30S RNA**, 50S RNA*, 50S RNA (5% g e l ) * , 50S RNA*, C s C l - t r e a t e d 50S RNA**, tRNA from sucrose the c o n t r o l (commercial E ^ c o l i  g r a d i e n t and  tRNA).  * Represents the t o t a l RNA from the 30S and 50S r i b o s o m a l subunits r e s p e c t i v e l y .  ** The 30S r i b o s o m a l  s u b u n i t was c e n t r i f u g e d through a C s C l  g r a d i e n t as d e s c r i b e d i n M a t e r i a l s and Methods and then the t o t a l RNA was i s o l a t e d from these described.  The 50S r i b o s o m a l  subunits  as p r e v i o u s l y  s u b u n i t was t r e a t e d  To f a c e page 76  similarly.  76  FIGURE 6 Sedimentation solution.  p a t t e r n o f E ^ c o l i ribosomal  The s u b u n i t s were prepared  subunits i n a CsCl  as d e s c r i b e d i n F i g u r e  4 and then c e n t r i f u g e d a t 36,000 rpm f o r 36 h r s a t 4° i n 61% (w/v) C s C l as d e s c r i b e d i n M a t e r i a l s and Methods. A. Represents the s e d i m e n t a t i o n (69A 6o 2  and  u n i t s ) through C s C l .  5 y l a l i q u o t s were removed f o r the absorbance  (483A 6o 2  u n i t s ) through C s C l .  subunits  One ml f r a c t i o n s were c o l l e c t e d  B. Represents the s e d i m e n t a t i o n  and  o f 30S r i b o s o m a l  readings.  o f 50S r i b o s o m a l  subunits  One ml f r a c t i o n s were c o l l e c t e d  5 y l a l i q u o t s were removed f o r the absorbance  readings.  To f a c e page 77  FIGURE 7 E l u t i o n p a t t e r n o f 100 mg o f commercial E ^ c o l i Sephadex G-100.  The RNA  was  column and e l u t e d w i t h 50 mM  chromatographed  tRNA from  on a 3 x 200  NaCl a t a flow r a t e o f 6 ml/hr.  F i v e ml f r a c t i o n s were c o l l e c t e d and one ml a l i q u o t s were removed f o r absorbance  cm  readings.  To face page 78  40  60  80 100 120 FRACTION NUMBER  140  160  180  200  79  subunit  shows o n l y one dense r e g i o n a t the top o f the g e l  i n d i c a t i n g t h a t i t c o n t a i n s o n l y high m o l e c u l a r The  weight  RNA.  g e l c o n t a i n i n g o n l y the RNA from the 50S ribosomal  subunit  a l s o has a dense r e g i o n a t the top o f the g e l b u t i n a d d i t i o n a double 5S RNA band w i t h what appears to be a t r a c e o f 4.5S and  4S RNA.  sucrose  The g e l c o n t a i n i n g the tRNA f r a c t i o n from the  g r a d i e n t a l s o shows a d i s t i n c t band i n the 5S r e g i o n .  The  s u b u n i t s were each put through a C s C l g r a d i e n t as  d e s c r i b e d i n M a t e r i a l s and Methods. sedimentation subunits.  d e n s i t y p a t t e r n o f each o f the C s C l - t r e a t e d  Each shows a s i n g l e peak.  s u b u n i t s were then e l e c t r o p h o r e s e d gel.  F i g u r e 6 shows the  The C s C l - t r e a t e d  i n a 10%  The r e s u l t s are shown i n F i g u r e 5.  polyacrylamide  The g e l c o n t a i n i n g  o n l y the RNA from C s C l - t r e a t e d 30S ribosomal  subunits  shows  a number o f bands from the top o f the g e l to the 5S RNA r e g i o n where there appears t o be a t r a c e band. ponding g e l c o n t a i n i n g the RNA somal s u b u n i t s and  The c o r r e s -  from C s C l - t r e a t e d 50S r i b o -  shows a s t r o n g 5S RNA band and d e f i n i t e  4.5S  4S RNA bands.  C h a r a c t e r i z a t i o n o f the Ribosomes F i g u r e 7 shows the o p t i c a l d e n s i t y p a t t e r n o f commercial E. c o l i tRNA  (General Biochemicals)  Sephadex G-100 column (200 x 3 cm). molecular  weight RNA,  f r a c t i o n a t e d on a long The f i r s t peak i s h i g h  the second peak 5S RNA and the t h i r d  peak, the major peak, c o n t a i n e d 8 shows a 10% p o l y a c r y l a m i d e  the 4S RNA m a t e r i a l .  Figure  g e l p a t t e r n o f peaks I I and I I I  FIGURE 8 Photograph o f p o l y a c r y l a m i d e  g e l (10%, pH 8.3 and 0.5 x 7 cm)  e l e c t r o p h o r e s i s p a t t e r n s o f d i f f e r e n t f r a c t i o n s o f commercial E. c o l i  tRNA chromatographed p r e v i o u s l y on a Sephadex  column (see F i g u r e 7 ) . III  From l e f t t o r i g h t  and the c o n t r o l (commercial E . c o l i  G-100  : peak I I , peak  tRNA).  To f a c e page 80  80  FIGURE 9 E l u t i o n p a t t e r n of t o t a l ribosomal E. c o l i on Sephadex G-100.  Tubes 1-25  amino a c i d a c c e p t o r  f r a c t i o n a t e d on a 3  50 mM NaCl a t a flow r a t e of  c o n t a i n 20 ml and a l l subsequent tubes  c o n t a i n 5 ml f r a c t i o n s . absorbance r e a d i n g s .  from  2  The RNA was  x 200 cm column and e l u t e d w i t h 6 ml/hr.  (8300A 6o u n i t s )  RNA  One ml a l i q u o t s were removed f o r  Solid  line  : A 6 o; d o t t e d 2  line  activity.  To f a c e page 81  : "*C1  FRACTION NUMBER  82  TABLE I I The  Peak  D i s t r i b u t i o n o f RNA i n E. c o l i Ribosomes  F r a c t i o n Number*  Total  A 6o 2  Units  % of Total  I  20-43  7897  97.2  II  45 - 64  128  1.5  65 - 120  103  1.3  III  The  f r a c t i o n s are those shown i n F i g u r e 9.  83  from F i g u r e 7.  The g e l s were run u s i n g an a l i q u o t from the  combined  f r a c t i o n s w i t h i n each peak r e g i o n .  The 4S  fraction  (peak I I I ) i s w e l l s e p a r a t e d from the 5S RNA  (peak II) a l t h o u g h i t does c o n t a i n a t r a c e o f 4.5S 5S RNA  RNA fraction  RNA.  The  f r a c t i o n a l s o c o n t a i n s the major p a r t o f the 4.5S  and a t r a c e o f 4S RNA o t h e r types o f RNA  material.  RNA  The reason t r a c e amounts o f  were found i n both peaks was because peak  tubes were not used i n the e l e c t r o p h o r e s i s r u n s .  This  will  be c l e a r l y demonstrated f u r t h e r on i n the t h e s i s . F i g u r e 9 shows the f r a c t i o n a t i o n o f the t o t a l o b t a i n e d from column. RNA, RNA  RNA  c o l i WRib run on the same Sephadex  G-100  The major peak i s h i g h m o l e c u l a r weight r i b o s o m a l  the second peak c o n t a i n s 5S rRNA and the t h i r d peak, 4S as c o n f i r m e d by a c c e p t o r s t u d i e s .  The l a s t peak con-  t a i n e d r e s i d u a l phenol l e f t over from the procedure used to o b t a i n the t o t a l r i b o s o m a l RNA and Methods. region.  The  which was  described i n Materials  T a b l e I I shows the amount o f RNA A260  r a t i o o f 4S  : 5S RNA  i s 0.87  i n each peak which  t h a t t h e r e are a p p r o x i m a t e l y 1.3 m o l e c u l e s o f 4S RNA the ribosomes per m o l e c u l e o f 5S RNA*.  indicates bound to  F i g u r e 10 shows the  s e p a r a t i o n o f peaks I I and I I I from F i g u r e 9 by e l e c t r o p h o r e s i s i n 10% p o l y a c r y l a m i d e g e l s .  The g e l s were run u s i n g an a l i q u o t  * If one assumes that one molecule of SS RNA is bound per ribosome and that in t h i s population of ribosomes one molecule of SS RNA represents I28A260 units, then the number of molecules of 4S RNA bound can be e a s i l y calculated given the molecular weight of SS and 4S RNA as 4 x 10 and 2.5 x IO respectively. 3  3  FIGURE 10 Photograph of p o l y a c r y l a m i d e g e l (10%, pH 8.3 and 0.5 x 7 cm) e l e c t r o p h o r e s i s p a t t e r n s of peaks I I and I I I as d e s i g n a t e d i n T a b l e I I . From l e f t t o r i g h t control  (commercial  E. c o l i  : peak I I , peak I I I and the  tRNA).  To f a c e page 84  84  85 TABLE I I I S p e c i f i c amino a c i d a c c e p t o r a c t i v i t y o f tRNA bound to E ^ c o l i  amino  acid  Ala Arg Asn Asp Gly Glu His He Leu Lys Met Phe Pro Ser Thr Trp Tyr Val  Ribosomal bound tRNA p moles/A 6 o unit 13.4 60.0 46 .4 43.4 16 .1 37.8 45.7 14.8 109 .4 108.5 209 .0 65.5 33.0 26.3 65.1 383.0 12.7 163.6 2  ribosomes  3  T o t a l tRNA* p moles/A2 6 o unit (19.0) (15.2)  106.9 (59.0) 73.6 42.7 (68.9) 72.3 (43.4) 27.3 43.6 (29 .3) (76.4)  Numbers o f known s p e c i f i c tRNAs i n ] 2 2 3 2 3 2 3 2 5 2 2 3 4 4 3 5 2 2  R e s u l t s shown i n b r a c h e t s were done i n t h i s l a b o r a t o r y R. Chase.  The o t h e r r e s u l t s i n t h i s column are those o f  B a r t z e t a l . (291) .  T o t a l RNA  r i b o s o m a l bound tRNA. References  by  274,  275.  See Appendix, page  163.  refers  to c y t o p l a s m i c and  FIGURE 11 E l u t i o n p a t t e r n o f the low m o l e c u l a r weight RNA  (1368A 6o  bound to E ^ c o l i ribosomes from Sephadex G-100.  The RNA  2  units) was  f r a c t i o n a t e d on a 3 x 200 cm column and e l u t e d w i t h 50 mM NaCl a t a flow r a t e of 6 ml/hr. collected. acceptor  Solid line  : A  2 6  o;  F i v e ml f r a c t i o n s were dotted l i n e  : '*C-amino 1  activity.  To face page 86  acid  86  FRACTION NUMBER  87  from the combined f r a c t i o n s shown i n T a b l e I I .  The  results  were i d e n t i c a l to those found i n s i m i l a r peaks i n F i g u r e  8.  The d o t t e d l i n e s i n F i g u r e 9 i n d i c a t e the r e g i o n of amino a c i d acceptor a c t i v i t y .  The peak tubes were pooled and  s t u d i e s were done on a l l the amino a c i d s except and c y s t e i n e .  The  acceptor  glutamine  r e s u l t s are shown i n T a b l e I I I .  E_^_ c o l i  ribosomes have bound tRNA which has a c c e p t o r a c t i v i t y the amino a c i d s . s p e c i e s to  The  amount of changing  v a r i e s from one  was  c a r r i e d out i n o r d e r to g e t more  d e f i n i t i v e d a t a on the d i s t r i b u t i o n o f the major low s p e c i e s bound t o ribosomes.  The  molecular  t o t a l r i b o s o m a l RNA  i s o l a t e d from WRib u s i n g the phenol  technique.  o f rRNA by t h i s procedure was  on the b a s i s o f the  A260  66.3%  The  u n i t s r e c o v e r e d from the s t a r t i n g m a t e r i a l .  d i s s o l v e d i n 100 mM  T r i s , pH  7.5  rRNA  and the s o l u t i o n was  made  made up 9% o f the t o t a l r i b o s o m a l RNA.  T h i s RNA  t a t e d w i t h c o l d ETOH, d i s s o l v e d i n 0.05  M NaCl,  The  column (3 x 200  cm)  and  The  The  was  RNA, precipi-  loaded i n t o a  The major peak  the second peak c o n t a i n e d  the minor peak, the 4S RNA  acceptor s t u d i e s .  as determined  by  d o t t e d l i n e s i n d i c a t e the r e g i o n of  amino a c i d a c c e p t o r a c t i v i t y .  The  was  and e l u t e d w i t h the same  r e s u l t s are shown i n F i g u r e 11.  c o n t a i n e d high m o l e c u l a r weight RNA, the 5S RNA  total  The  s u p e r n a t a n t , which c o n t a i n e d the low m o l e c u l a r weight  Sephadex G-100  was  recovery  2 M w i t h r e s p e c t to NaCl and kept a t 4° f o r a day.  buffer.  tRNA  another.  Another experiment  RNA  for a l l  recovery of RNA  from  the  88  TABLE IV D i s t r i b u t i o n o f low m o l e c u l a r weight RNA i n E. c o l i ribosomes i s o l a t e d by Sephadex G-100  Fraction  Total A 6o units 2  chromatography*  % of Total  A c t u a l % bound t o ribosomes  Peak I  656  50.7  95.5  Peak I I  404  31.2  2.8  Peak I I I  230  18.1  1.7  * The d a t a are from F i g u r e  11.  FIGURE 12 Photograph o f p o l y a c r y l a m i d e g e l (10%, pH 8.3 and 0.5 x 7 cm) e l e c t r o p h o r e s i s p a t t e r n s o f the peak f r a c t i o n o f each peak r e g i o n o f F i g u r e 11.  From l e f t  to r i g h t  f r a c t i o n 116, f r a c t i o n 132, f r a c t i o n s control  (commerical  E. c o l i  : fraction  86,  116 and 132 and the  tRNA).  b  To f a c e p a g e 89  89  FIGURE 12a Chromoscan t r a c i n g  o f a mixture  of f r a c t i o n s  (peaks I I and I I I ) from F i g u r e 11. both  116 and 132  Equal c o n c e n t r a t i o n s of  f r a c t i o n s were mixed.  To f a c e page 90  91 column was  95%.  T a b l e IV shows t h e d i s t r i b u t i o n o f RNA  t h e s e peak r e g i o n s . a p p r o x i m a t e l y 0.58 m a t e l y 0.90 o f 5S RNA*.  The  A 6o 2  r a t i o o f 4S : 5S RNA  in  was  : 1 which i n d i c a t e s that there i s a p p r o x i -  m o l e c u l e o f 4S bound t o the ribosomes p e r m o l e c u l e The peak f r a c t i o n i n each peak r e g i o n o f F i g u r e  11 was r u n on a 10% p o l y a c r y l a m i d e g e l . shown i n F i g u r e 12.  The r e s u l t s a r e  Peak I ( F r a c t i o n 86) c o n t a i n e d one  band w h i c h remained a t the top o f the g e l and had a m o l e c u l a r w e i g h t i n e x c e s s o f 6S.  major  presumably  Peak I I ( f r a c t i o n  116)  c o n t a i n e d 3 major bands - a d o u b l e 5S RNA band and the  4.5S  RNA  band w h i l e peak I I I ( f r a c t i o n 132)  c o r r e s p o n d i n g t o 4S RNA. 116 and 132  c o n t a i n e d the bands  Equal concentrations of  (peaks II and I I I ) were combined and  fractions electro-  phoresed i n a 10% p o l y a c r y l a m i d e g e l ( F i g u r e 1 2 ) . was  This g e l  s u b s e q u e n t l y scanned i n a J o y c e - L o e b l Chromoscan.  r e s u l t s are shown i n F i g u r e 12a. d o u b l e band.  The 4S RNA appears as a probably  The m a t e r i a l p r e c e d i n g 4S RNA  W i t h o u t t a k i n g the degraded RNA  The  is^degraded  i n t o a c c o u n t , the 4.5S  r e p r e s e n t s 2.7% o f the t o t a l RNA.  RNA. RNA  Even though t h i s 4.5S  i s s p r e a d t h r o u g h o u t t h e 4 t o 5S RNA  RNA  region, i t s t i l l repre-  s e n t s much l e s s t h a n one m o l e c u l e p e r m o l e c u l e o f 5S  RNA.  O t h e r T e c h n i q u e s used t o F r a c t i o n a t e Low M o l e c u l a r W e i g h t  RNA  (a) P r e p a r a t i v e G e l E l e c t r o p h o r e s i s Commercial  E_^ c o l i tRNA (5 mg)  was  f r a c t i o n a t e d i n a 10%  * If one assumes that one molecule of 5S RNA is bound per ribosome and that in t h i s population of ribosomes one molecule of 5S RNA represents 404Az6o units, then the number of molecules of 4S RNA bound can be e a s i l y calculated given the molecular weights of 5S and 4S RNA as 4 x 10 and 2.5 x 10 respectively. 3  3  FIGURE 13 Electrophoretic  p a t t e r n i n a 10% p r e p a r a t i v e  g e l o f 5 mg o f commercial E_^ c o l i  tRNA.  The  procedure i s g i v e n i n M a t e r i a l s and Methods. 60 ml/hr and 5 ml f r a c t i o n s were c o l l e c t e d . were removed f o r absorbance  polyacrylamide fractionation Flow r a t e was One ml  readings.  To f a c e page 9 2  fractions  92  I  L_  0  I  10  I  20  I  30  I  40  FRACTION NUMBER  l  50  I  60  FIGURE 14 Electrophoretic  p a t t e r n o f 100 mg o f commercial E ^ c o l i  i n a 10% p r e p a r a t i v e p o l y a c r y l a m i d e  gel.  tRNA  The f r a c t i o n a t i o n  procedure i s g i v e n i n M a t e r i a l s and Methods.  To f a c e page 9 3  93  FIGURE 15 Chromatography o f 100 mg o f commercial E ^ c o l i Sephadex A-50.  tRNA on DEAE-  The RNA was e l u t e d w i t h a 0.45-0.60 M NaCl  gradient buffered  i n 20 mM T r i s , pH 7.6.  The flow r a t e was  20 ml/hr and 3.2 ml f r a c t i o n s were c o l l e c t e d .  To face page 9 4  94  A  P  !u  P  Oi O  P  In  260nm P  P  i>  O  NaCl GRADIENT  FIGURE 16 Photograph o f p o l y a c r y l a m i d e  g e l (10%, pH 8.3  and 0.5 x 7  cm)  e l e c t r o p h o r e s i s p a t t e r n s o f the peak f r a c t i o n o f each peak r e g i o n o f F i g u r e 15. I),  f r a c t i o n 113  control  From l e f t t o r i g h t  (peak I I ) , f r a c t i o n 173  (commercial E . c o l i  : f r a c t i o n 90  (peak  (peak IV) and the  tRNA).  To f a c e page 95  95  polyacrylamide  g e l u s i n g a Canalco p r e p a r a t i v e g e l e l e c t r o -  p h o r e s i s apparatus as d e s c r i b e d i n M a t e r i a l s and Methods. r e s u l t s are shown i n F i g u r e 13.  A l i q u o t s from the peak  f r a c t i o n s were then e l e c t r o p h o r e s e d gel. RNA  The major peak c o n t a i n e d and a t r a c e o f 5S RNA,  l a s t peak, 5S RNA.  i n a 10%  4S RNA,  polyacrylamide  the second peak, 4S  the t h i r d peak, 4.5S RNA  from the peak f r a c t i o n s were then e l e c t r o p h o r e s e d  5S RNA,  gel.  Peak I c o n t a i n e d  peak I I c o n t a i n e d  o f 4.5S RNA o f 4S and 5S  and the  F i g u r e 14 shows the f r a c t i o n a t i o n on a  much l a r g e r s c a l e where 100 mg o f tRNA were used.  polyacrylamide  The  5S RNA,  Aliquots i n a 10%  4S RNA w i t h a t r a c e o f  4S RNA w i t h t r a c e amounts  and 5.8S RNA w h i l e peak I I I c o n t a i n e d  t r a c e amounts  RNA.  (b) DEAE-Sephadex Chromatography. Commercial E ^ c o l i tRNA  (100 mg) was loaded onto a DEAE-  Sephadex A-50 column (0.9 x 120 cm) p r e v i o u s l y e q u i l i b r a t e d w i t h 0.45 M NaCl b u f f e r e d i n 20 mM T r i s , pH 7.6. w i t h a 0.45-0.60 M NaCl g r a d i e n t  (283).  The tRNA was e l u t e d  The g r a d i e n t was  checked r a d i o m e t r i c a l l y by use o f a c o n d u c t i v i t y meter. r e s u l t s are shown i n F i g u r e 15.  The  The o p t i c a l d e n s i t y p a t t e r n i s  almost i d e n t i c a l t o t h a t i n F i g u r e 13.  The peak f r a c t i o n s were  electrophoresed  g e l and the r e s u l t s are  i n a 10% p o l y a c r y l a m i d e  shown i n F i g u r e 16. and  some 4.5S RNA,  Peak I c o n t a i n s v i r t u a l l y a l l the 4S peak I I c o n t a i n s  w h i l e peak IV c o n t a i n s o n l y 5S  5S RNA and some 4S  RNA  RNA  RNA.  S i n c e the p r e p a r a t i v e g e l e l e c t r o p h o r e s i s gave a much b e t t e r s e p a r a t i o n o f 4S RNA  than the DEAE-Sephadex A-50, i t  FIGURE 17 Electrophoretic A260  weight RNA  (19 40  u n i t s ) from E_^ c o l i ribosomes i n a 10% p r e p a r a t i v e  poly-  acrylamide  gel.  p a t t e r n o f the low m o l e c u l a r  The f r a c t i o n a t i o n procedure i s g i v e n i n  M a t e r i a l s and Methods. were c o l l e c t e d .  Flow r a t e 60 ml/hr and 3 ml  Solid line :  A 6o; 2  dotted  fractions  l i n e : ^C-amino 1  a c i d acceptor a c t i v i t y .  To f a c e page 9 7  FRACTION NUMBER  FIGURE 18 Photograph o f p o l y a c r y l a m i d e electrophoretic  35  and 0.5  x 7  cm)  p a t t e r n s of the peak f r a c t i o n o f each peak  r e g i o n o f F i g u r e 17. fraction  g e l (10%, pH 8.3  From l e f t  to r i g h t  (peak I I ) , f r a c t i o n 70  (commercial E. c o l i  : fraction  26  (peak I I I ) And the  tRNA).  To f a c e page 9 8  (peak I ) , control  FIGURE 19 Spectrum o f f r a c t i o n 26 f i r s t chromatographed was  (peak I) of F i g u r e 17.  on Whatman No.  run on the e l u t e d spots  An a l i q u o t  1 paper and a spectrum  (A and B) as d e s c r i b e d i n the  Results.  To f a c e page 99  was  99  100 was  used t o c a r r y out the f r a c t i o n a t i o n o f r i b o s o m a l  F r a c t i o n a t i o n of Ribosomal RNA  By P r e p a r a t i v e G e l  RNA.  Electro-  phoresis The  total  low m o l e c u l a r  weight r i b o s o m a l  as d e s c r i b e d i n M a t e r i a l s and Methods and preparative g e l . dotted The  The  separated  obtained  in a  l i n e s i n d i c a t e r e g i o n s of amino a c i d a c c e p t o r  main absorbance peak d i d not correspond  i n a 10%  r e s u l t s are shown i n F i g u r e 18.  4S RNA,  and  a t r a c e amount of 5S RNA.  c o n t a i n s 4S RNA  and  10%  The activity.  t o the main peak  A l i q u o t s from the f r a c t i o n s of peak  absorbance were e l e c t r o p h o r e s e d  5S RNA  was  r e s u l t s are shown i n F i g u r e 17.  of acceptance a c t i v i t y .  The  RNA  some 5S RNA,  and  The  polyacrylamide f i r s t peak  The  gel.  contains  second peak  the l a s t peak  contains  only.  E f f o r t s were made to c h a r a c t e r i z e the main peak s i n c e i t was  a r e g i o n of c o m p a r a t i v e l y  (a) An No.  low  amino a c i d a c c e p t o r  a l i q u o t from the main peak was  1 paper and  s p o t t e d onto Whatman  the paper chromatogram was  run f o r 16 hours a t  room temperature i n a s o l v e n t system c o n s i s t i n g of acid  : concentrated  obtained, migrated  one  NH<,OH:H20 (66:1:33).  a t the o r i g i n and  beyond any  Heppel's technique spots.  The  Two  spots were  (ADP,  ATP  GDP,  GTP).  The  e l u t e d o f f the paper w i t h H 2 O u s i n g  (284).  A spectrum was  run on the e l u t e d  r e s u l t s are shown i n F i g u r e 19.  T r a c i n g A,  f l u o r e s c e n t s p o t does not show the t y p i c a l RNA t r a c i n g B,  isobutyric  a f l u o r e s c e n t spot which  o f the standards  spots were c u t o u t and  activity  the  trace while  the s p o t a t the o r i g i n , shows a more " t y p i c a l "  RNA  FIGURE The  effect  Figure  17.  20  o f pH o n t h e s p e c t r u m o f f r a c t i o n A i s a t pH  1 1 . 0 ; B a t pH  7.0;  26  (peak I )  C a t pH  To f a c e p a g e  1.0.  101  101  102  trace.  (b) A spectrum of the peak f r a c t i o n a t v a r i o u s  v a l u e s was  run and  appears to be i n pH  the r e s u l t s are shown i n F i g u r e  very l i t t l e  a t l e a s t as  pH  20.  There  change i n the spectrum w i t h changes  f a r as the  X max  and  X min  are  concerned,  (c) A s m a l l amount o f the peak f r a c t i o n (7.2A2 6 0 u n i t s ) put on a s m a l l D E A E - c e l l u l o s e column (0.5 w i t h an  (NHi»)  u n i t s ) was and  an  gradient.  2CO3  eluted  X min  The  The  pH  I t had  M  was  s i m i l a r t o t h a t of t r a c i n g B, F i g u r e  a p p r o x i m a t e l y 50% sample was or 1.0  N KOH.  f r a c t i o n was and  9.6.  show a t y p i c a l RNA  (NHi»)  254  A  19.  spectrum. There was  of the sample unaccounted f o r .  eluted with 2 M  of  e l u t e d w i t h approximately  1.9  2  eluted  an X max  of the s o l u t i o n was  minor f r a c t i o n (0.5A2 6 0 u n i t s ) was (NHi») C03 but d i d not  and  p r i n c i p a l f r a c t i o n (2.8A260  i n the v o i d volume.  of 244.  x 6 cm)  was  2CO3  No  It still  further  i n 7 M u r e a , 0.1  N  KOH  (d) Approximately 28A260 u n i t s of the peak put  on a DEAE-Sephadex A-50  e l u t e d w i t h a NaCl g r a d i e n t  buffer containing  7Murea a t pH  (0.45 7.4.  column (0.9  -1.0  M)  x 32  i n 20 mM  A s i n g l e peak was  cm)  Tris eluted  i n the v o i d volume which showed an absorbance t r a c i n g s i m i l a r to the p r i n c i p a l f r a c t i o n of approximately The  Recovery  was  74%.  p r i n c i p a l f r a c t i o n s i n (c) and  phoresed i n a 10% c o u l d be  (c) above.  obtained.  p o l y a c r y l a m i d e g e l but  (d) were e l e c t r o no d e f i n i t i v e r e s u l t s  A t the p r e s e n t moment the  of the main peak remains to be e s t a b l i s h e d .  characterization Besides  i n g some tRNA, the p r i n c i p a l peak must c o n t a i n RNA  of  containlower  FIGURE 21 Growth and pH curves o f an L, c o l i p y r i m i d i n e - r e q u i r i n g (ATCC 13135).  The d e s c r i p t i o n o f the growth medium i s g i v e n  i n M a t e r i a l s and Methods. line  mutant  Solid  line  : c e l l growth, d o t t e d  : pH.  Symbols :  •  •  growth a t 4 ug/ml  •  •  growth a t 300 ug/ml  • •  — • pH a t 4 ug/ml  uracil uracil  uracil  • pH a t 300 ug/ml  uracil  The arrow i n d i c a t e s a d d i t i o n o f one ml u r a c i l  (300 ug/ml).  To face page 10 3  1 0 3  10 4  molecular  weight s i n c e presumably t h i s would be e l u t e d  from the p r e p a r a t i v e g e l .  T h i s low m o l e c u l a r  first  weight RNA may  j u s t be degraded RNA and the presence o f such a h i g h  concen-  t r a t i o n o f t h i s m a t e r i a l may be due t o the nature o f the p r e p a r a t i v e g e l procedure.  T h i s may be one o f the p i t f a l l s o f  the procedure and t h i s technique  was abandoned i n f a v o r o f the  demonstrated s u p e r i o r i t y o f Sephadex G-100 as a means o f fractionating  low m o l e c u l a r  weight RNA.  Exchange Experiments An E ^ c o l i p y r i m i d i n e - r e q u i r i n g mutant (ATCC 13135) was grown i n a m i n i m a l - s a l t s medium i n the presence o f 4 ug u r a c i l / m l as d e s c r i b e d i n M a t e r i a l s and Methods. shows the growth curve concentrations.  F i g u r e 21  o f the mutant a t two d i f f e r e n t  uracil  When the c e l l s were grown i n the lower  c o n c e n t r a t i o n o f u r a c i l , c e l l growth proceeded normally and then l e v e l l e d o f f .  A d d i t i o n o f the h i g h e r c o n c e n t r a t i o n o f  u r a c i l r e s u l t e d i n an immediate marked i n c r e a s e i n c e l l growth. When the c e l l s were grown i n the h i g h e r u r a c i l , c e l l growth a l s o proceeded normally off.  concentration of and then  levelled  The a d d i t i o n o f the same c o n c e n t r a t i o n o f u r a c i l had no  f u r t h e r e f f e c t on c e l l The  growth.  pH o f the medium decreased  d u r i n g c e l l growth b u t  remained c o n s t a n t d u r i n g the s t a t i o n a r y p e r i o d s r e g a r d l e s s o f the c o n c e n t r a t i o n o f u r a c i l i n the medium. Once the growth p a t t e r n o f t h i s mutant s t r a i n was e s t a -  FIGURE 22 Displacement o f l a b e l l e d tRNA bound t o E^_ c o l i ribosomes. E. c o l i mutant d e s c r i b e d i n F i g u r e 21 was used.  The  The  p r e p a r a t i o n o f the ribosomes i s d e s c r i b e d i n M a t e r i a l s and Methods. Symbols  : Tube 1  •  •  s t r i p p e d tRNA + ATA  Tube 2  A —  A  aa-tRNA + PM  Tube 3  •  • s t r i p p e d tRNA + ATA + PM  Tube 4  *d  aa-tRNA + ATA + PM  Conditions of displacement: At  0 wash each tube c o n t a i n e d i n a t o t a l volume o f  10 ml, 2 mg o f the p a r t i c u l a r u n l a b e l l e d tRNA and 150A260 u n i t s WRib b u f f e r e d i n 10 mM T r i s , 10 mM Mg(OAc) , pH 7.6. 2  Tubes 1, 3 and 4 were made 70 uM w i t h r e s p e c t t o ATA and tubes 2, 3, and 4 ImM w i t h r e s p e c t t o PM. . Tubes 1 and 2 were i n c u b a t e d f o r 30 mins a t 0° and tubes 3 and 4 f o r 30 mins a t 30°*. A f t e r the second wash each tube was i n c u b a t e d a t 30° i n the presence o f 10 mg o f u n l a b e l l e d tRNA. A f t e r the f o u r t h wash each tube was i n c u b a t e d a t 24° f o r 20 mins i n a s o l u t i o n c o n t a i n i n g 0.5 mM GTP, 3 mM ATP, 50 mM NH^Cl, 750yl S100, 400ul S100A and 10 mg o f u n l a b e l l e d tRNA. A f t e r the f i f t h wash each tube was i n c u b a t e d a t 24° for  20 mins i n a b u f f e r c o n t a i n i n g 50 mM T r i s ,  pH 7.6 and 10 mg o f u n l a b e l l e d  tRNA.  5 mM Mg(OAc)2/  FIGURE 22 - c o n t i n u e d A f t e r the s i x t h wash each tube was i n c u b a t e d a t 37° f o r 30 mins i n a s o l u t i o n c o n t a i n i n g 50 mM KC1 and 10 mg o f unlabelled  tRNA. A f t e r the seventh wash each tube was i n c u b a t e d a t  0° w i t h 0.1 mM Mg(OAc) b u f f e r e d a t pH 7.3 ( b u f f e r K) and 2  10 mg o f u n l a b e l l e d  tRNA.  * A f t e r each i n c u b a t i o n p e r i o d , the WRib o b t a i n e d by c e n t r i f u g a t i o n were resuspended i n 10 ml o f s o l u t i o n c o n t a i n i n g the p a r t i c u l a r components f o r d i s p l a c e m e n t .  To f a c e page 105  105  106  b l i s h e d the c e l l s were grown i n the same m i n i m a l - s a l t s  medium  but  milli-  t h i s time 8 ug u r a c i l / m l o f medium was  c u r i e of u r a c i l - 6 - H  (9.16  3  cells.  The  harvested.  ug)  was  c e l l s were grown to the  added.  One  a l s o added to l a b e l  the  l a t e l o g phase and  then  Ribosomes were prepared i n the u s u a l manner  were found to be very (Table I ) .  active i n a protein-synthesizing  A preliminary  the c o n d i t i o n s  experiment was  c a r r i e d out  and system  to study  necessary to d i s p l a c e the l a b e l l e d tRNA bound  to these ribosomes.  The  r e s u l t s are shown i n F i g u r e  22.  Displacement appears to be v i r t u a l l y complete a f t e r s i x washes r e g a r d l e s s o f the c o n d i t i o n s I t was  decided  same manner as was  used.  to c a r r y out an exchange experiment i n done by Cannon e t al.  (148) .  In  the  this  experiment, a b u f f e r system i s employed under c e r t a i n c o n d i t i o n s which enables exchange between u n l a b e l l e d tRNA  and  l a b e l l e d r i b o s o m a l bound tRNA to r e a d i l y take p l a c e . WRib were mixed i n a b u f f e r c o n t a i n i n g Mg  + +  a t pH  7.4.  Unlabelled  B i o c h e m i c a l s ) was  added  (15x  c a l c u l a t e d to be p r e s e n t incubated  a t 0-4°  on  c o l i B tRNA  a l i q u o t was had  10  mM  (General  the WRib) and  the mixture  was  f o r 30 mins w i t h o c c a s i o n a l s t i r r i n g .  The  H-  p e l l e t was  After  c e n t r i f u g e d a t 105,000  mixed w i t h f r e s h b u f f e r ,  an  removed to determine the amount o f exchange t h a t  taken p l a c e  incubation  Tris,  3  the amount of l a b e l l e d tRNA  the i n c u b a t i o n p e r i o d the mixture was x g for 5 hrs.  10 mM  The  and  repeated.  then u n l a b e l l e d tRNA was The  added and  washings were repeated  until  the  10 7  TABLE V Exchange o f l a b e l l e d tRNA bound t o E. c o l i  No. o f washings  ribosomes w i t h u n l a b e l l e d  Ribosomal p e l l e t * * (cpm)  tRNA  N o n - p e l l e t e d ribosomes* (cpm)  0  33884  1  25809  57187  2  23548  4663  3  18333  31026  4  15080  16968  5  14974  3579  6  13728  1047  * The n o n - p e l l e t e d ribosomes were p u t through a M i l l i p o r e filter the  (1.2 y ) .  filter  (290).  Ribosomes w i t h bound tRNA remained on The f i l t e r was d r i e d and counted i n a  toluene s c i n t i l l a t i o n mix. ** The r i b o s o m a l p e l l e t was mixed w i t h 10 ml o f b u f f e r (see Text) and a 5 y l a l i q u o t was counted i n a dioxane t i o n mix.  scintilla-  FIGURE 23 Exchange o f l a b e l l e d tRNA bound to E ^ c o l i u n l a b e l l e d tRNA.  ribosomes w i t h  Ribosomes were prepared as mentioned i n  F i g u r e 22 from the E ^ c o l i mutant d e s c r i b e d i n F i g u r e  21.  D e t a i l s o f the exchange c o n d i t i o n s are g i v e n i n the R e s u l t s .  To f a c e page 10 8  10 8  NUMBER OF WASHINGS i  109  exchange was  complete  (Table V and F i g u r e 23).  According  t o the t a b l e a g r e a t number of counts were found i n the supernatant  ( n o n - p e l l e t e d ribosomes).  The extremely h i g h l e v e l s  of counts i n some o f the supernatants was the presence of some ribosomes centrifugation. complete  p r o b a b l y due t o  r e s u l t i n g from incomplete  Although exchange appeared t o be  virtually  a f t e r f o u r washes, two more exchanges were c a r r i e d  out u n t i l the l o s s of counts t o the s u p e r n a t a n t was T h i s minimal l o s s was  minimal.  a c h i e v e d by c e n t r i f u g i n g the r i b o s o m a l  mixture f o r extended p e r i o d s of time t o ensure t h a t a l l o f the ribosomes had been p e l l e t e d .  These ribosomes were found  t o be i n a c t i v e i n a p r o t e i n - s y n t h e s i z i n g  system.  F o l l o w i n g the exchange experiment, the t o t a l r i b o s o m a l RNA  was  p r e p a r e d from these H-WRib as d e s c r i b e d i n M a t e r i a l s  and Methods. b u f f e r e d a t pH  3  T h i s RNA 7.6  was  d i s s o l v e d i n 10 mM  ( b u f f e r F) which was  w i t h r e s p e c t t o NaCl.  then made two  The h i g h m o l e c u l a r weight RNA  ted immediately and was  molar precipita-  c o l l e c t e d by c e n t r i f u g a t i o n w h i l e the  low m o l e c u l a r weight RNA h i g h m o l e c u l a r weight RNA  remained was  r e p r e c i p i t a t e d i n 2 M NaCl. the p r e v i o u s one.  Mg(OAc)2  i n the supernatant.  The  r e d i s s o l v e d i n b u f f e r F and The supernatant was  Sodium a c e t a t e (1.5 M, pH 5.2)  combined w i t h was  added  t o g i v e a 2% s o l u t i o n and the low m o l e c u l a r weight RNA  was  subsequently p r e c i p i t a t e d by the a d d i t i o n o f 3 volumes of cold ethanol.  T h i s low m o l e c u l a r weight RNA  Sephadex G-100  column  (200 x 3 cm)  was  put on a  and e l u t e d w i t h 50 mM  NaCl.  FIGURE Elution pattern weight RNA  24  from Sephadex G-100 o f the low m o l e c u l a r  (2000A2 6 0 u n i t s ) bound t o l a b e l l e d  a f t e r exchange w i t h u n l a b e l l e d tRNA. exchange c o n d i t i o n s a r e d e s c r i b e d  coli  ribosomes  The ribosomes and the  i n Figure  23.  The  RNA  was f r a c t i o n a t e d on a 3 x 200 cm column and e l u t e d w i t h 50 mM NaCl a t a flow r a t e o f 6 ml/hr.  F i v e ml f r a c t i o n s were  collected.  To face page 110  FRACTION NUMBER  FIGURE 25 Photograph o f p o l y a c r y l a m i d e  g e l (10%, pH 8.3 and 0.5 x 7 cm)  e l e c t r o p h o r e s i s p a t t e r n s o f v a r i o u s f r a c t i o n s from F i g u r e From l e f t  24.  t o r i g h t : f r a c t i o n s 85, 106, 116, 121, 125, 130,  135, 142 and the c o n t r o l  (commercial E . c o l i  tRNA).  To face page 111  Ill  112 TABLE VI D i s t r i b u t i o n o f l a b e l l e d H-RNA 3  i n E ^ c o l i ribosomes a f t e r with unlabelled  Fraction high molecular RNA  exchange  tRNA  cpm/A 6o 2  weight 19465  5S  RNA  2707  4S  RNA  55  unit  113  The  r e s u l t s a r e shown i n F i g u r e  high molecular  weight RNA w h i l e  p a r t o f the RNA as i n d i c a t e d . and  24. The f i r s t peak  contained  the r e s t c o n t a i n the major A l i q u o t s o f the peak f r a c t i o n s  a number o f f r a c t i o n s between the second and t h i r d peaks  were e l e c t r o p h o r e s e d  i n 10% p o l y a c r y l a m i d e  a r e shown i n F i g u r e 25. The f i r s t peak  gels.  The r e s u l t s  ( f r a c t i o n 85) a l s o  c o n t a i n s a s m a l l amount o f 5.8S and 5S RNA.  The second peak  ( f r a c t i o n 116) c o n t a i n s o n l y a t r a c e o f 4S RNA w h i l e the t h i r d peak ( f r a c t i o n 142) c o n t a i n s v i r t u a l l y and to  some 4.5S RNA.  The  a l l the 4S RNA  r a t i o o f 4S : 5S RNA had i n c r e a s e d  A260  almost 3.5 i n ribosome exchanged w i t h u n l a b e l l e d tRNA.  T h i s i s comparable t o about 5.7 molecules o f 4S RNA t o one molecule o f 5S RNA*.  I n untreated  ribosomes the r a t i o o f 4S :  5S RNA on a t o t a l RNA b a s i s v a r i e d from 0.58 (Table IV) to 0.90 (Table I I ) .  T h i s amounted t o 1-2 molecules o f tRNA bound p e r  molecule o f 5S RNA; a v a l u e which compared very the l i t e r a t u r e  favourably  with  (286-289).  A l i q u o t s o f the 3 peaks f r a c t i o n s i n F i g u r e 24 were taken and  the amount o f l a b e l l e d H-RNA was determined. 3  are shown i n T a b l e V I . had  The r e s u l t s  I t appears t h a t almost 100% exchange  taken p l a c e between u n l a b e l l e d tRNA and the l a b e l l e d tRNA  o r i g i n a l l y bound t o the ribosomes. * If one assumes that one molecule of SS RNA is bound per ribosome and that in t h i s population of ribosomes one molecule of SS RNA represents the t o t a l amount i s o l a t e d in ^ 2 6 0 units, then the number of molecules of 4S RNA bound can be e a s i l y calculated given the molecular weights of 5S and 4S RNA as 4 x 10 and 2.5 x 10 respectively. 3  3  FIGURE 26 Chromatography on B D - c e l l u l o s e  o f 4S RNA  (140A 6o units) 2  to E_^ c o l i ribosomes a f t e r exchange w i t h u n l a b e l l e d  bound  tRNA.  F r a c t i o n s 135-170 (Figure 24) were f r a c t i o n a t e d on a column (100 x 0.9 cm) w i t h the i n d i c a t e d NaCl c o n t a i n i n g  10 mM M g C l  The flow r a t e was  2  (dashed l i n e ) g r a d i e n t of  i n a t o t a l volume o f one  42 ml/hr and 5 ml f r a c t i o n s were c o l l e c t e d .  A t the i n d i c a t e d p o i n t , e l u t i o n was continued ethanol  gradient  liter.  i n 1 M NaCl c o n t a i n i n g  t o t a l volume o f 400 ml.  with a  10 mM M g C l  The flow r a t e was  2  0-30% in a  42 ml/hr and 5 ml  f r a c t i o n s were c o l l e c t e d .  To face page  114  114  MOLARITY OF NaCl  FIGURE 27 Chromatography on B D - c e l l u l o s e  o f 4S RNA  n o r m a l l y bound to E ^ c o l i ribosomes. 11) were f r a c t i o n a t e d on a column indicated MgCl  2  (140A 6 0 u n i t s ) 2  F r a c t i o n s 126-140  (112 x 0.9  cm) w i t h the  (dashed l i n e ) g r a d i e n t o f NaCl c o n t a i n i n g  i n a t o t a l volume o f one l i t e r .  (Figure  The flow  10  mM  r a t e was  42  ml/hr and 5 ml f r a c t i o n s were c o l l e c t e d .  A t the i n d i c a t e d  p o i n t , e l u t i o n was  ethanol  continued  1 M NaCl c o n t a i n i n g The flow r a t e was  10 mM  w i t h a 0-30%  MgCl  2  gradient i n  i n a t o t a l volume o f 400 ml.  42 ml/hr and 5 ml f r a c t i o n s were c o l l e c t e d .  To face page  115  116  The  acrylamide  g e l a n a l y s i s ( F i g u r e 25)  f r a c t i o n s from the Sephadex G-100 t h a t f r a c t i o n s 135-170 c o n t a i n e d some 4.5S  RNA  column ( F i g u r e 24) i n d i c a t e d v i r t u a l l y a l l the 4S RNA  b u t were d e v o i d o f 5S RNA.  were put on a B D - c e l l u l o s e column (100 e q u i l i b r a t e d with The  RNA  was  0.35  a (0.35-1.0 M)  liter.  T h i s was  e t h a n o l g r a d i e n t i n 1 M NaCl and o f t h i s g r a d i e n t was 26.  x 0.9  fractions  cm)  previously  400  ml.  The  Mg . + +  NaCl g r a d i e n t i n a  f o l l o w e d by a  10 mM  and  MgCl2.  0-30%  T o t a l volume  r e s u l t s are shown i n F i g u r e  Four peak r e g i o n s were i s o l a t e d i n the NaCl g r a d i e n t  although  the second peak r e g i o n i s i t s e l f  3 peaks.  The  a c t u a l l y be  bound to E ^ c o l i  ribosomes.  p a t t e r n shown i n F i g u r e 26. there i s a s h o u l d e r .  weight RNA  of and  Figure  normally  I t i s somewhat d i f f e r e n t from I n p l a c e of the f i r s t peak  the  (Figure  There i s a sharp, w e l l - d e f i n e d second  a broader t h i r d peak which shows a s m a l l peak on i t s  t r a i l i n g edge. b e t t e r separated Figure  d e f i n i t e peak  a second component.  27 shows the p a t t e r n o f low m o l e c u l a r  peak and  a c t u a l l y made up  e t h a n o l g r a d i e n t c o n t a i n s one  a s h o u l d e r which may  26)  These  M NaCl i n the presence of 10 mM  e l u t e d with  t o t a l volume o f one  of various  The  peaks i n the e t h a n o l g r a d i e n t were much  than i n the p r e v i o u s  experiment  (Figure  26).  28 shows the o p t i c a l d e n s i t y p a t t e r n o b t a i n e d when  commercial Ej_ c o l i  tRNA was  s a l t g r a d i e n t e l u t e d two  d e f i n i t e peaks, the second having  s l i g h t shoulder which may component.  The  ethanol  f r a c t i o n a t e d on B D - c e l l u l o s e .  The a  i n d i c a t e the p o s s i b i l i t y of a t h i r d  f r a c t i o n c o n t a i n e d o n l y one  component  FIGURE Chromatography The  RNA  1.5  cm)  ( 3 6 5 0 A 6 o u n i t s ) was with  At  the i n d i c a t e d  10 mM  f l o w r a t e was  MgCl  2  of commercial E^ c o l i  10 mM  (106  (dashed l i n e ) g r a d i e n t  of NaCl  i n a t o t a l volume o f t h r e e  liters.  x  90 m l / h r a n d 20 m l f r a c t i o n s w e r e c o l l e c t e d .  t h e i n d i c a t e d p o i n t , e l u t i o n was  containing  tRNA.  f r a c t i o n a t e d on a c o l u m n  2  containing The  on B D - c e l l u l o s e  28  MgCl  2  i n 10%  (v/v)  continued with  1.10  ethanol.  To f a c e p a g e  117  NaCl  FRACTION NUMBER  118  but t h i s may have been due to the f a c t that a gradient was not employed. Aliquots were taken from every second f r a c t i o n  (Figure  26) to see i f the l a b e l l e d tRNA that remained bound to  coli  ribosomes a f t e r the exchange with unlabelled tRNA was spread evenly throughout the e l u t i o n pattern, or whether i t was concentrated i n a c e r t a i n region.  I t was  found to be spread  uniformly throughout the fractions eluted with the NaCl gradient. unit.  The r a d i o a c t i v i t y amounted to 25-50 cpm per  A260  In the ethanol gradient the r a d i o a c t i v i t y reached a  maximum l e v e l of 250 cpm per  A260  unit.  An aliquot of the peak  f r a c t i o n was electrophoresed i n a 10% polyacrylamide gel and was found to contain some 4.5S  RNA.  This may  high counts i n t h i s p a r t i c u l a r f r a c t i o n . i s that according to Table I I I , t R N A  Trp  account for the  Another p o s s i b i l i t y  i s bound to WRib i n  the highest amount and therefore i t may not be as r e a d i l y exchanged as the other tRNAs. i n the ethanol f r a c t i o n the high counts. plausible  Since tRNA ^ i s found s o l e l y Tr  (295) i t therefore could account for  The former explanation seems to be more  since there i s no reason to believe that one p a r t i -  cular tRNA i s less vulnerable to exchange than another.  119 DISCUSSION The carried  experiments d e s c r i b e d i n t h e p r e v i o u s o u t i n an attempt t o c l a r i f y  the b i o l o g i c a l  the low m o l e c u l a r weight RNAs a s s o c i a t e d w i t h somes.  In p a r t i c u l a r ,  coli  i d e n t i f i e d b u t i n most cases  c o u l d n o t be e s t a b l i s h e d .  evidence  E_j_ c o l i  Nevertheless,  Some o f these  RNAs  their function a method was found  ribosomes d e v o i d o f a l l 4S components b u t no  was o b t a i n e d  f o r a s p e c i f i c 4S f r a c t i o n which might  f u n c t i o n during p r o t e i n synthesis i n the chain mechanism.  ribo-  ribosomes which had  undergone e x t e n s i v e washing p r o c e d u r e s .  for preparing  role of  these experiments were designed t o  c h a r a c t e r i z e the RNA bound t o  were c l e a r l y  s e c t i o n s were  This r e s u l t helps  the non-existence  t o confirm  of a chain-terminating  termination  r e c e n t reports- on tRNA.  The mechanism  of c h a i n t e r m i n a t i o n i n p r o t e i n b i o s y n t h e s i s i s s t i l l an unsolved  problem.  Present  evidence  suggests t h a t t h e r e may  be t h r e e nonsense o r t e r m i n a t i n g codons, UAA, UAG and UGA. According  t o t h e Wobble H y p o t h e s i s  a l s o code f o r Cys and T r p .  (30 7 ) , however, UGA may  No s u p p r e s s o r  which suppresses only ochre mutants  has y e t been found  (UAA), although  suppres-  sors e x i s t which suppress both ochre and amber mutants S i n c e UAA has been found t o be t h e t e r m i n a t o r  (UA ). 0  codon f o r a l l  the phage p r o t e i n s thus f a r i d e n t i f i e d , i t i s p o s s i b l e t h a t at l e a s t one s p e c i f i c t e r m i n a t i n g tRNA c o u l d be i n v o l v e d . However, the groups i n v e s t i g a t i n g t h i s problem c o u l d not demonstrate such a tRNA s p e c i e s i n t h e t e r m i n a t i o n  step.  Most o f t h e i n f o r m a t i o n about t h e mechanism o f c h a i n  120  t e r m i n a t i o n was  o b t a i n e d by t h e use o f two  I n one o f t h e s e , C a p e c c h i (43, 75)  assay systems..  used as mRNA, the  from a mutant R17 phage, i n w h i c h the s e v e n t h c o a t p r o t e i n gene was  viously  (p. 2 9 ) .  codon i n t h e  a nonsense codon (UAG).  of t h e h e x a p e p t i d y l - t R N A The  was  RNA  The  formation  c a r r i e d out as d e s c r i b e d  r e s u l t i n g hexapeptidyl-tRNA  a t t a c h e d t o the mRNA-ribosome complex.  The  pre-  remained  release of free  h e x a p e p t i d e from t h i s complex depended on a p r o t e i n component, designated supernatant  r e l e a s e f a c t o r (R f a c t o r ) from the h i g h speed (S100) o f  coli.  Bretscher  same assay s y s t e m , i n c u b a t e d the S100 a l k a l i n e c o n d i t i o n s and s u b s e q u e n t l y  (84) , u s i n g  supernatant  under m i l d  treated i t with perio-  d a t e t o d e s t r o y the amino a c i d a c c e p t o r a b i l i t i e s o f endogenous tRNAs.  the  the  He t h e n added o n l y t h o s e aa-tRNA s p e c i e s  needed t o form the h e x a p e p t i d e p l u s the p e r i o d a t e - t r e a t e d supernatant  and got  Nirenberg's  chain-termination.  group ( 8 8 ) , u s i n g a AUG•UAA•ribosome complex  as the t e r m i n a t i o n a s s a y , was  a b l e t o get c h a i n  termination  t o t a k e p l a c e upon a d d i t i o n o f t h e c r u d e R f a c t o r . subsequently  found t h a t t h e R f a c t o r was  They  a c t u a l l y two  ent enzymes w i t h d i f f e r e n t codon s p e c i f i c i t i e s  (89,  differ-  91).  Another p r o t e i n , c a l l e d S f a c t o r , which served to c a t a l y z e the t e r m i n a t i o n r e a c t i o n , was Recently  also isolated  I s h i t s u k a and K a j i  (109)  (90, 9 2 ) .  i s o l a t e d a TR f a c t o r  (tRNA r e l e a s e f a c t o r ) which they s u g g e s t e d worked hand w i t h t h e R f a c t o r - - t h e R f a c t o r h y d r o l y z e d  hand-in-  the e s t e r  l i n k between t h e p e p t i d e and the tRNA w h i l e TR d i s p l a c e d the  121 tRNA from i t s s i t e on the ribosome. Each o f the groups working i n t h i s t h e i r discussions that t h e i r results evidence  f o r the nonexistence  f i e l d stipulated i n  were not c o n c l u s i v e  of a chain-terminating  tRNA.  In c o n t r a s t t o the case o f i n i t i a t i o n where a s p e c i f i c  tRNA  is d e f i n i t e l y  tRNA  i n v o l v e d , the requirement f o r a s p e c i f i c  i n t e r m i n a t i o n has n o t , as y e t , been shown b u t the p r e s e n t understanding required.  o f the problem i s t h a t such a tRNA i s not  The c h a i n - t e r m i n a t i n g  i n which h i g h l y p u r i f i e d  experiments o u t l i n e d above  tRNAs were used  l e s s were not p r o p e r l y c o n t r o l l e d  (84, 88), neverthe-  s i n c e i t can be argued t h a t  the t e r m i n a t i n g tRNA c o u l d have remained bound t o the r i b o somes.  These p r e v i o u s  i n v e s t i g a t o r s f a i l e d t o show t h a t  t h e i r ribosomes o r r i b o s o m a l  s u b u n i t s were d e v o i d o f 4S  RNA.  T h i s tRNA c o u l d occupy a s i t e on the ribosome d i s t i n c t from the normal tRNA s i t e s .  Thus, t h i s s p e c i f i c tRNA o r tRNA-  l i k e component c o u l d be an i n t e g r a l Previous  p a r t o f the ribosome.  i n v e s t i g a t o r s s t u d i e d the p r o t e i n s i n v o l v e d  i n t h e t e r m i n a t i o n mechanism and from t h e i r r e s u l t s  concluded  t h a t t h e p o s s i b l e involvement o f a s p e c i f i c RNA component was remote.  T h i s r e p o r t i s the f i r s t known d i r e c t study o f  the RNA bound t o ribosomes w i t h  respect to i t s possible  involvement i n c h a i n - t e r m i n a t i o n . The  f o l l o w i n g q u e s t i o n s which have y e t t o be posed  concerning  chain-termination  c o u l d be asked:  (1) What i s the nature o f the low molecular a s s o c i a t e d w i t h p u r i f i e d ribosomes?  weight  RNA  122  (2)  Is t h e r e a s p e c i f i c low m o l e c u l a r weight RNA o t h e r than 5S RNA which cannot be e q u i l i b r a t e d w i t h tRNA?  (3)  I f such an RNA ination?  e x i s t s i s i t i n v o l v e d i n c h a i n term-  In o r d e r to b e g i n  i t was  neces-  s a r y t o o b t a i n a c l e a n ribosome p r e p a r a t i o n which was  active  in protein synthesis. for  the study  ized,  Ribosomes from  f o r t h r e e reasons:  and  (c) a l l p r e v i o u s  been c a r r i e d out w i t h  (a) they  are w e l l  chain-termination  these ribosomes.  r e p o r t e d i n the l i t e r a t u r e . Nirenberg's (WRib) and The  group had  The  studies  i n these s t u d i e s w i t h  had (c)  of the p r o j e c t  followed.  ribosomes used i n a l l experiments to be from  coli B cells  discussed  grown to the  or l a t e l o g phase (see M a t e r i a l s and Methods).  various concentrations  M NHi»Cl  washing w i t h 0.5  ribosomal  RNase I (29 6-29 8) .  7.4.  not t i g h t l y bound  A number of i n v e s t i g a t o r s had  prolonged  results  0.5-1.0  mid-  The  of magnesium b u f f e r e d at pH  These steps removed a l l enzymes (and RNA) ribosomes.  those  r e p o r t e d the b e s t ribosome p r e p a r a t i o n  p r e p a r a t i o n i n v o l v e d seven i n c u b a t i o n s w i t h  to  be  l a t t e r reason  At the b e g i n n i n g  t h e i r procedure was  were r e a d i l y prepared  at  character-  of p a r t i c u l a r importance s i n c e i t would be e a s i e r t o  c o r r e l a t e the r e s u l t s o b t a i n e d  log  c o l i were chosen  (b) the ease w i t h which a c t i v e ribosomes c o u l d  obtained,  was  t o answer the q u e s t i o n s  a l s o shown t h a t  M NHJ.C1 d e a c t i v a t e d o r removed Others have found v a r i a b l e  i n t h a t some of the RNase a c t i v i t y had been removed  by NHi»Cl washing b u t the ribosomes s t i l l cant l e v e l of RNase (29 8) .  retained a  Ribosomal a c t i v i t y was  signifidetermined  123 by  f o l l o w i n g the uptake of  phenylalanine  ll,  C-Phe i n a polyU-dependent  i n c o r p o r a t i o n system.  The  a c t i v i t y o f the  pre-  p a r a t i o n s v a r i e d between 50-100 f o l d over the c o n t r o l o r background l e v e l  (see R e s u l t s , p. 68).  The  ribosomes were 67%  as c o n t r a s t e d w i t h the u s u a l v a l u e of 60-6 3% a d d i t i o n a l c o n f i r m a t i o n of the h i g h p u r i t y The  (6) which  RNA  was  obtained.  c h o i c e of the p u r i f i c a t i o n procedure w i l l ,  of  course,  depend on the use t o which the ribosomes are to be p u t ,  the  nature o f the i m p u r i t i e s which are t o be  Often  a b a l a n c e must be  removed, e t c *  found between the p u r i f i c a t i o n and  h a n d l i n g of the ribosomes s i n c e h a n d l i n g may logical activity. of p u r i t y .  destroy  In g e n e r a l , t h e r e i s no a b s o l u t e  Each p r e p a r a t i o n must be  over-  judged by  their biostandard  an o p e r a t i o n a l  criterion. Keeping the l a t t e r statement i n mind, a development o c c u r r e d a t a s t a g e when the p r o j e c t was advanced.  Iwasaki e t a l . (219)  already  r e p o r t e d a new  considerably  and very  quick  method o f o b t a i n i n g very a c t i v e ribosome p r e p a r a t i o n s . method e s s e n t i a l l y  The  i n v o l v e d washings i n 1 M NH..C1 f o l l o w e d by  e l u t i o n through a D E A E - c e l l u l o s e  column (RSI, Chart I I ) .  a c t i v i t y o f these f r e s h ribosome p r e p a r a t i o n s was 1 5 0 - f o l d over the c o n t r o l or background l e v e l .  The  approximately  Because of  the  advanced s t a g e of the p r e s e n t p r o j e c t , t h i s l a t t e r method of p r e p a r i n g ribosomes was necessary  not adopted s i n c e i t would have been  t o r e p e a t a l l the experiments a l r e a d y completed.  t h i s reason  and  t i o n i n use was  those a l r e a d y mentioned, the ribosome  For  prepara-  deemed s u i t a b l e f o r the experiments i n which  124 i t would be The  employed.  a c t i v i t y o f the ribosome p r e p a r a t i o n s was  v a r i a b l e as shown i n T a b l e I . activity  l e v e l s was  quite  Perhaps the d i f f e r e n c e i n  caused by v a r i a t i o n s i n h a n d l i n g  the work-up o f the r i b o s o m a l p r e p a r a t i o n .  during  I t i s w e l l known  t h a t ribosomes which are q u i t e s t a b l e at low  temperature  and  i o n i c s t r e n g t h become u n s t a b l e when e i t h e r the temperature or i o n i c s t r e n g t h i s r a i s e d o r the c o n c e n t r a t i o n o f M g - i o n s i s ++  reduced.  Under the former c o n d i t i o n s , the ribosomes were  found by numerous i n v e s t i g a t o r s t o have l a t e n t r i b o s o m a l  RNase  I a c t i v i t y w h i l e the l a t t e r c o n d i t i o n s tended t o a c t i v a t e the enzyme (298).  F o l l o w i n g such treatments  which d i s r u p t the  s t r u c t u r e o f the ribosome, the enzyme, i f p r e s e n t , i s a b l e t o a t t a c k both  the r i b o s o m a l  RNA  and  added f r e e RNA  under c o n d i -  t i o n s o f temperature, i o n i c s t r e n g t h and M g - i o n + +  where i n t a c t ribosomes showed no RNase a c t i v i t y The  ribosomal preparations  I I I ) were two The  concentration  (298).  used i n Table I (RSI, I I  and  month o l d WRib which had been s t o r e d at - 7 0 ° .  o r i g i n a l f r e s h p r e p a r a t i o n had  an a c t i v i t y  than the b l a n k which c o n t a i n e d no ribosomes  82-fold greater  (Table I ) .  i n v e s t i g a t o r s have i n d i c a t e d t h a t t h e i r r i b o s o m a l remained a c t i v e f o r up t o t h r e e weeks  (219)  Most  preparations  w h i l e others have  o b t a i n e d a c t i v e p r e p a r a t i o n s w i t h s i x month o l d p r e p a r a t i o n s (218).  Although  Nirenberg  (218)  was  able to f r e e z e and thaw  h i s WRib p r e p a r a t i o n s s e v e r a l times w i t h o u t  undue l o s s  of  a c t i v i t y , the WRib p r e p a r a t i o n used i n experiment I I , Table was  almost i n a c t i v e a f t e r f r e e z i n g and  thawing t w i c e .  This  I  125  may have been due t o t h e f a c t t h a t t h e p r e p a r a t i o n was a l r e a d y two months o l d .  The r e a s o n f o r our i n a b i l i t y t o m a i n t a i n  a c t i v e p r e p a r a t i o n s o v e r e x t e n d e d p e r i o d s o f t i m e may  also  have been t h e r e s u l t o f t h e method used t o f r e e z e t h e p r e p a r a tions .  They were q u i c k l y f r o z e n i n d r y i c e p r i o r t o s t o r a g e  a t -70°. liquid N  On t h e o t h e r hand N i r e n b e r g f r o z e h i s e x t r a c t s i n 2  p r i o r to storage i n l i q u i d N  2  refrigerators.  though t h e p r e p a r a t i o n s were k e p t a t -70° t h e p o s s i b l e o f some n u c l e a s e a c t i v i t y cannot be d i s c o u n t e d .  Even presence  For i n s t a n c e ,  S z e r (299) , u s i n g an RNase I ~ s t r a i n o f E ^ c o l i , found  almost  complete d i s a p p e a r a n c e o f 23S RNA w i t h t h e c o n c o m i t a n t  forma-  t i o n o f 16S RNA i n t h e l a r g e r r i b o s o m a l s u b u n i t a t 0°.  The  f i n a l p r o d u c t s s u g g e s t e d t h e i n v o l v e m e n t o f an RNase I V found i n t h e RNase I " s t r a i n .  RNase I I , w h i c h i s a l s o p r e s e n t i n  t h i s s t r a i n , l o s e s 9 0% o f i t s a c t i v i t y a f t e r 24 h r s when k e p t c o l d o r f r o z e n (300).  However, when our " o l d " WRib p r e p a r a -  t i o n s were p a s s e d t h r o u g h a DE-22 column and e l u t e d w i t h 1 M NHi»Cl, t h e o r i g i n a l a c t i v i t y o f t h e WRib, a t t h e p a r t i c u l a r c o n c e n t r a t i o n u s e d , was r e e s t a b l i s h e d  (RSI, Table I , Chart I I ) .  A s i m i l a r phenomenon was r e c e n t l y r e p o r t e d by Scheps e t a l . (26 8) who o b s e r v e d t h a t t h e a c t i v i t y o f a r i b o s o m a l e x t r a c t c o u l d be r e s t o r e d by a t e m p e r a t u r e dependent p r e i n c u b a t i o n i n t h e p r e s e n c e o f 0.56 M NH^Cl f o l l o w e d by i n c u b a t i o n i n 1 M NHi,Cl.  They s u g g e s t t h a t t h i s i s due t o a d e c r e a s e i n t h e  amount o f 70S ribosomes w h i c h were found t o have i n c r e a s e d i n t h e f o r m e r l y i n a c t i v e p r e p a r a t i o n s . They a l s o found t h a t t h e d e f e c t i n v o l v e d b o t h s u b u n i t s and was n o t due t o an i n a c t i v e  126 S100  which c o n t a i n s the s y n t h e t a s e a c t i v i t y .  I t should  also  be borne i n mind t h a t as mentioned e a r l i e r , washing ribosomes w i t h 0.5 RNase I  M NH4CI tends (296-298).  t o remove some of the r i b o s o m a l bound  But Ochoa's group  two p r o l o n g e d washings w i t h 0.5  (219)  found t h a t a f t e r  M NH^Cl the ribosomes  c o n t a i n e d a h i g h l e v e l o f RNase a c t i v i t y .  still  However, when the  ribosomes were chromatographed on a D E A E - c e l l u l o s e column the RNase a c t i v i t y was  reduced by about 9 9%.  active i n protein synthesis.  T h i s may  These ribosomes were  account  f o r the very  a c t i v e WRib p r e p a r a t i o n s which were o b t a i n e d when the WRib were p r e p a r e d by washing through Table I ) . experiments  The  a DE-22 column (RSI, Chart I I ,  reason f o r not u s i n g t h i s p r e p a r a t i o n i n a l l  was  already discussed.  These o b s e r v a t i o n s show  t h a t RNase can be removed from E ^ c o l i ribosomes w i t h o u t i n g t h e i r i n a c t i v a t i o n ; the enzyme normally t o be bound very f i r m l y t o the ribosomes.  caus-  appears, however, Unfortunately,  none of the f r e s h r i b o s o m a l p r e p a r a t i o n s used i n the p r e s e n t s t u d i e s was  checked  f o r the presence  of RNase a c t i v i t y ,  although they were e x t e n s i v e l y t r e a t e d w i t h were not subsequently I t may  e l u t e d through  and  1 M NHi»Cl they  a D E A E - c e l l u l o s e column.  be r e a s o n a b l e t o assume t h a t s i n c e these RNases are  normally so t i g h t l y bound t o the ribosomes, activity  a t l e a s t some RNase  remained a s s o c i a t e d w i t h them (WRib).  I t i s a l s o known t h a t as a b a c t e r i a l c u l t u r e e n t e r s l a t e l o g a r i t h m i c and s t a t i o n a r y phases i t s a b i l i t y e x t r a c t active i n s y n t h e s i z i n g peptides decreases. been observed  f o r s e v e r a l b a c t e r i a l species  (218,  the  to y i e l d T h i s has 266,  267)  an  127 and i s seen as w e l l w i t h n a t u r a l mRNA and i n the p o l y U - d i r e c t e d s y n t h e s i s of p o l y p h e n y l a l a n i n e .  To compare the a c t i v i t y  of  c e l l - f r e e p r o t e i n - s y n t h e s i z i n g e x t r a c t s from d i f f e r e n t l o t s  of  c e l l s , the c e l l s s h o u l d be h a r v e s t e d i n the same p h y s i o l o g i c a l s t a t e , a c o n d i t i o n which i s o f t e n d i f f i c u l t t o o b t a i n ment I I , T a b l e I ) .  The  c e l l s used i n a l l the  (experi-  experiments  except the exchange s t u d i e s were o b t a i n e d commercially batches.  One  the o t h e r was experiments  batch was  i n two  h a r v e s t e d a t the mid-log phase w h i l e  h a r v e s t e d a t the l a t e l o g phase.  The  exchange  were c a r r i e d out w i t h c e l l s grown i n the  presence  of l a b e l l e d u r a c i l and h a r v e s t e d i n the l a t e l o g t o s t a t i o n a r y phase.  Although WRib prepared  from each b a t c h of c e l l s were  very a c t i v e i n p r o t e i n s y n t h e s i s , the a c t i v i t y per v a r i e d from b a t c h t o b a t c h and t h i s may  A260  have been due,  unit i n part,  t o the stage a t which the c e l l s had been h a r v e s t e d . F i g u r e 1 shows the s e p a r a t i o n o f low m o l e c u l a r weight  RNA  from the d i f f e r e n t ribosome p r e p a r a t i o n s (Chart I I , Table I) a f t e r e l e c t r o p h o r e s i s i n a 10% p o l y a c r y l a m i d e g e l . appears  t o be a marked d i f f e r e n c e i n the amount of tRNA bound  t o ribosomes which have been put through column and subsequently  aggregates  a DEAE-cellulose  e l u t e d w i t h 1 M NH^Cl.  q u i t e l i k e l y t h a t the column has  a way  It  appears  not only removed r i b o s o m a l  but a l s o l o o s e l y bound tRNA which may  on the ribosomes, i n such  There  and/or a l t e r the conformation  block s i t e s of the ribosome  as t o p r e v e n t the normal tRNA exchange  from  taking place. Once an a c t i v e ribosome p r e p a r a t i o n was  obtained experi-  128 t o prepare WRib d e v o i d o f a l l 4S com-  ments were undertaken  ponents w h i l e a t the same time m a i n t a i n i n g the a c t i v i t y o f the ribosome.  V a r i o u s methods were used t o remove bound tRNA  The r e s u l t s shown i n F i g u r e 2 i n d i c a t e d t h a t puro-  ribosomes. mycin  (PM)  T h i s was  from  treatment  alone d i d not remove a l l the bound tRNA.  expected s i n c e PM occupies o n l y one s i t e on the  some, the p e p t i d y l s i t e .  ribo-  Aminoacyl-tRNA bound t o the a c c e p t o r  s i t e does not r e a c t w i t h PM  (269,  have shown t h a t w h i l e PM reduced  285) .  (285)  K u r i k i and K a j i  the amount of bound p e p t i d y l -  tRNA, i t d i d not a l t e r the amount of ribosome-bound tRNA. the absence of s o l u b l e enzymes and GTP, of ribosomes,  tRNA and peptidyl-tRNA  the i s o l a t e d  w i t h the h y p o t h e s i s f o r peptidyl-tRNA  complex  bound a d d i t i o n a l tRNA  s u g g e s t i n g t h a t the ribosome c o n t a i n e d two s i t e s tRNA and one s i t e f o r p e p t i d y l - t R N A .  In  f o r aminoacyl-  These d a t a are c o n s i s t e n t  t h a t d u r i n g p o l y p e p t i d e s y n t h e s i s the  site  and one s i t e f o r aminoacyl-tRNA are c o n s t a n t l y  o c c u p i e d b u t the o t h e r s i t e f o r aminoacyl-tRNA i s o c c u p i e d transiently. both s i t e s  Only i n the absence of p e p t i d e bond f o r m a t i o n are  f o r aminoacyl-tRNA c o n s t a n t l y o c c u p i e d .  A t any  time, 50% of the t o t a l bound tRNA s h o u l d be d i s p l a c e d by and t h i s would account observed  i n Figure 2 .  f o r the decreased These PM-treated  one  PM  amount o f bound tRNA WRib were a l s o  found  to be more a c t i v e than the o r i g i n a l p r e p a r a t i o n ; a r e s u l t t h a t i s i n agreement w i t h t h a t observed by Scheps e t a l . (26 8 ) . They suggested to polyU was tide chains.  t h a t the i n a b i l i t y  due PM  o f the ribosome t o  respond  to t h e i r b e i n g b l o c k e d by u n f i n i s h e d polypepovercame t h i s by d i s p l a c i n g the  peptidyl-tRNA,  129 c a u s i n g t h e r e l e a s e o f growing p e p t i d e  chains.  D i a l y s i s o f PM-treated WRib a g a i n s t low M g the bound tRNA ( F i g u r e 2 ) .  + +  removed a l l  T h i s was t o be expected s i n c e the  removal o f t h e nascent p o l y p e p t i d e  c h a i n by PM would remove  the s t a b i l i z i n g e f f e c t i t e x e r t e d on the b i n d i n g o f t h e tRNA to  t h e ribosome.  be c o n v e r t e d  Under these c o n d i t i o n s t h e ribosomes would  t o t h e i r subunits  i n v e s t i g a t o r s that ribosomal material  (265,  that ribosomal  270, 271). subunits  when t r e a t e d f i r s t w i t h ribosomal  subunit  decreased  subunits  are d e v o i d o f 4S RNA  Other i n v e s t i g a t o r s have suggested  are only d e v o i d o f 4S RNA m a t e r i a l PM b u t i n the absence o f PM t h e 50S  c o n t a i n s peptidyl-tRNA  tunately the d i a l y s i s inactive.  and i t has been found by some  a g a i n s t low M g  + +  (115,  272).  Unfor-  l e f t the WRib v i r t u a l l y  T h i s may have been due t o the f a c t t h a t t h e Mg'*"'" c o n c e n t r a t i o n d e s t a b i l i z e d the n u c l e o p r o t e i n  s t r u c t u r e r e s u l t i n g i n the a c t i v a t i o n o f the l a t e n t  ribosomal  RNase I (29 8) . D i a l y s i s o f t h e WRib a g a i n s t low M g (0.1 mM) removed v i r t u a l l y Cannon e t a l . (148)  + +  ion  a l l t h e bound tRNA (Figure 3 ) .  have found t h a t i n the absence o f p r o t e i n  s y n t h e s i s o r mRNA, and a t 0.1 mM M g  + +  ion concentration, the  ribosomes were d i s s o c i a t e d i n t o t h e i r s u b u n i t s ensuing  process  washed o f f . E. c o l i ,  concentration  and i n t h e  a l l t h e bound tRNA was shown t o be completely  A f t e r protein synthesis i n a c e l l - f r e e extract o f  a s m a l l f r a c t i o n o f the tRNA t h a t was bound t o the  ribosomes i n h i g h M g  + +  i o n c o n c e n t r a t i o n became r e s i s t a n t t o  b e i n g washed o f f i n low M g  + +  i o n concentrations.  According t o  130 Cannon e t a l . t h i s amounted t o about h a l f a m o l e c u l e per ribosome.  o f tRNA  T h i s had been i n t e r p r e t e d as due t o t h e p r e s e n c e  o f a n a s c e n t p o l y p e p t i d e c h a i n on t h e tRNA w h i c h the b i n d i n g o f t h e tRNA t o t h e r i b o s o m e .  stabilized  As was f o u n d i n t h e  p r e v i o u s e x p e r i m e n t s w i t h PM, d i a l y s i s a g a i n s t low magnesium n o t o n l y removed a l l 4S RNA b u t a l s o l e f t t h e ribosomes virtually inactive.  Once a g a i n , t h e r e a s o n  f o r the i n a c t i v e  p r e p a r a t i o n s may have been due t o n u c l e a s e a c t i o n . R i b o s o m a l s u b u n i t s were p r e p a r e d ribosomes by c e n t r i f u g a t i o n through results  from these d i a l y z e d  a sucrose g r a d i e n t .  ( F i g u r e 5) showed t h a t t h e r e was v i r t u a l l y no 4S  The RNA  m a t e r i a l bound t o t h e 50S r i b o s o m a l s u b u n i t and a b s o l u t e l y none bound t o t h e 30S r i b o s o m a l s u b u n i t . quent C s C l treatment  Although  the subse-  o f t h e s e s u b u n i t s s h o u l d have removed o n l y  the s p l i t p r o t e i n s l e a v i n g b e h i n d t h e 23S and 40S subunits  ribosomal  ( 6 0 ) , i t appears t h a t some d e g r a d a t i o n must have  o c c u r r e d t o a c c o u n t f o r t h e a d d i t i o n a l bands o b s e r v e d  i n Figure  5; f o r example, a 5S RNA band i n t h e 30S p a r t i c l e and a 4S RNA band i n t h e 50S p a r t i c l e .  I t has been shown t h a t i n t h e  absence o f mRNA, 30S. p a r t i c l e s do n o t have a f f i n i t y  f o r tRNA  and t h a t b i n d i n g i s s p e c i f i c f o r t h e 50S r i b o s o m a l s u b u n i t s . I n t h e p r e s e n c e o f messenger t h e r e i s a l s o s p e c i f i c b i n d i n g t o the 30S p a r t i c l e .  G i l b e r t has shown t h a t t R N A - l i n k e d  nascent  p o l y p h e n y l a l a n i n e remains a t t a c h e d t o t h e 50S p a r t i c l e even a f t e r complete d i s s o c i a t i o n o f t h e ribosomes i n t o (263).  subunits  E l s e n (264, 2 6 5 ) , on t h e o t h e r hand, o b s e r v e d  the  r e l e a s e o f 4S RNA m a t e r i a l from t h e 50S p a r t i c l e i n t h e p r e s e n c e  131 of h i g h  salt.  Attempts were made t o o b t a i n an a c t i v e r i b o s o m a l p r e p a r a t i o n u s i n g the i n d i v i d u a l s u b u n i t s i n s t e a d of whole WRib. s u b u n i t s , i n s t e a d of WRib, were used  The  i n the assay system f o r  d e t e r m i n i n g p o l y p h e n y l a l a n i n e s y n t h e s i s as d e s c r i b e d i n M a t e r i a l s and Methods. s u b u n i t was  Twice as much of the 50S  added t o the system as 30S  r i b o s o m a l s u b u n i t be-  cause of the d i f f e r e n c e s i n m o l e c u l a r weight r i b o s o m a l a c t i v i t y was c o n t a i n e d no ribosomes.  found t o be The  variations  (9 3) .  The  j u s t above the b l a n k which  f o l l o w i n g reasons  the f a i l u r e t o a c h i e v e r i b o s o m a l a c t i v i t y : were p r e p a r e d  ribosomal  may  account f o r  (a) the s u b u n i t s  from 2-month o l d WRib and t h i s t o g e t h e r w i t h  i n h a n d l i n g d u r i n g the work-up and p r e p a r a t i o n o f  s u b u n i t s may  have i n a c t i v a t e d the p r e p a r a t i o n , (b) the p r e p a r a -  t i o n of r i b o s o m a l s u b u n i t s i n v o l v e d d i a l y s i s a g a i n s t low magnesium which i n t u r n causes  the d e s t a b i l i z a t i o n of the  n u c l e o p r o t e i n s t r u c t u r e r e s u l t i n g i n the a c t i v a t i o n o f the l a t e n t r i b o s o m a l RNase I a c t i v i t y .  T h i s RNase has been  t o be l o c a t e d e x c l u s i v e l y on the 30S  subunit  (298).  found  However,  i t has been e s t i m a t e d t h a t no more than one  ribosome i n about  t e n would c a r r y a molecule  Szer  that i f freshly of L  coli  o f RNase (29 8 ) .  i s o l a t e d 70S  found  ribosomes from an RNase I " s t r a i n  are f r a c t i o n a t e d i n t o s u b u n i t s and kept a t 0 ° , the  d e g r a d a t i o n goes f u r t h e r and both The  (299)  f i n a l products suggested  23S  and 16S  the involvement  found i n t h i s RNase I " s t r a i n .  RNAs are h a l v e d . of an RNase IV  The e f f e c t of RNases on whole  WRib as compared t o t h e i r s u b u n i t s w i l l be d i s c u s s e d l a t e r ,  132 (c) the c o n d i t i o n s used t o observe r i b o s o m a l were not o p t i m a l .  subunit  activity  For i n s t a n c e , the r a t i o of Mg /ATP w i l l ++  determine the degree of attachment of the amino a c i d s t o tRNAs  (27 8).  WRib may  The  r a t i o which i s o p t i m a l i n a system c o n t a i n i n g  not be o p t i m a l i n the same system c o n t a i n i n g the  u n i t s i n s t e a d of WRib. (159)  the  According  t o Pestka  the a c t i v i t y o f d i f f e r e n t 30S  and  ribosomal  Nirenberg  subunit  prepara-  t i o n s v a r i e d and  70S  ribosomes formed by r e a s s o c i a t i o n of  purified  50S  ribosomal  30S  and  sub-  s u b u n i t s were only about h a l f  as a c t i v e i n b i n d i n g aa-tRNA as n o n - d i s s o c i a t e d  70S  ribosomes.  I t s h o u l d be noted here t h a t a f t e r the above experiments had been completed Tompkins e t a l . (93) ribosomes prepared work and w i t h  prepared  subunits  i n an i d e n t i c a l manner as i n the  present  these s u b u n i t s , they were able t o o b t a i n  a c t i v e ribosomal  preparation.  T h i s p r e p a r a t i o n was  from  an  also active  i n c h a i n t e r m i n a t i o n u s i n g the Caskey t e r m i n a t i o n assay  (88).  S i n c e our s u b u n i t s  and  contained  n e g l i g i b l e amounts o f tRNA  s i n c e t h i s t e r m i n a t i o n assay c o n t a i n e d (fMet-tRNA) i t may  be  only i n i t i a t o r  assumed, i n d i r e c t l y , t h a t the  t e r m i n a t i o n mechanism does not r e q u i r e a s p e c i f i c tRNA.  T h i s p o i n t w i l l be  tRNA  chain-  terminating  c l a r i f i e d f u r t h e r on i n the  thesis.  A t t h i s stage i n the development o f the t h e s i s the f o l l o w i n g p o i n t s had been e s t a b l i s h e d : (a) a method f o r prep a r i n g a c t i v e ribosomes , (b) treatment o f WRib w i t h  PM  removed  some of the bound tRNA l e a v i n g the p r e p a r a t i o n more a c t i v e than the o r i g i n a l , and  (c) d i a l y s i s  the ribosomes i n t o s u b u n i t s  a g a i n s t low magnesium d i s s o c i a t e d  d e v o i d of 4S RNA  and  inactive i n  133 protein synthesis. N i r e n b e r g was ribosomes  a b l e t o get c h a i n - t e r m i n a t i o n u s i n g whole  and s i n c e a c t i v e ribosomes  i t was  f e l t t h a t the proposed  weight  RNA,  c o u l d be r e a d i l y  s t u d i e s of the low  prepared  molecular  p a r t i c u l a r l y the tRNA bound to ribosomes,  c o u l d be  c a r r i e d out w i t h these p r e p a r a t i o n s . As mentioned e a r l i e r the WRib used i n a l l the may  experiments  have c o n t a i n e d some nucleases as e v i d e n c e d by the  t h a t ribosome a c t i v i t y  fact  c o u l d be r e s t o r e d by e l u t i o n through  D E A E - c e l l u l o s e column which i s known t o remove RNases The same e f f e c t c o u l d have been due aggregates  t o the removal  a  (281).  of r i b o s o m a l  or tRNA which b l o c k e d s i t e s on the ribosome and i n  so doing p r e v e n t e d the normal tRNA exchange from t a k i n g p l a c e . P r e v i o u s evidence suggested t h a t n u c l e a s e a c t i v i t y on WRib remained  l a t e n t p r o v i d e d the v a r i a t i o n s  p r e p a r a t i o n of WRib were minimized  i n h a n d l i n g d u r i n g the  and t h e r e f o r e t h i s was  only p r e c a u t i o n taken t o e l i m i n a t e n u c l e a s e a c t i v i t y . experiments  the  Since  were c a r r i e d out o n l y w i t h a c t i v e p r e p a r a t i o n s ,  c o n t r o l experiments  t o determine  the n u c l e a s e a c t i v i t y o f these  p r e p a r a t i o n s were c o n s i d e r e d unnecessary.  Most o f the p e r t i -  nent d a t a r e c o r d e d i n the l i t e r a t u r e a l s o tended t o suggest t h a t tRNA bound t o whole ribosomes i n s t a n c e , Cannon e t a l . (148) ribosomes  was  was  RNase r e s i s t a n t .  found t h a t tRNA bound t o  r e s i s t a n t t o p a n c r e a t i c RNase.  found t h a t i n the presence o f ribosomes was  s u b u n i t s i n response  70S  (162)  and p o l y U , Phe-tRNA  s u b s t a n t i a l l y p r o t e c t e d from h y d r o l y s i s .  of t h i s tRNA t o 30S  Pestka  For  Although b i n d i n g  t o polyU was  subs tan-  134 tial,  the tRNA was not p r o t e c t e d from p a n c r e a t i c RNase d i g e s -  tion.  The presence o f both s u b u n i t s was r e q u i r e d f o r  s u b s t a n t i a l p r o t e c t i o n o f t h e tRNA from RNase d i g e s t i o n .  The  d a t a a l s o i n d i c a t e d t h a t the Phe-tRNA which was bound t o the ribosomes  and r e s i s t a n t t o RNase remained i n t a c t , t h a t i s , the  aminoacyl end o f the aminoacyl-tRNA some (149 , 255).  Neu and Heppel  was p r o t e c t e d by the r i b o -  (301) found t h a t r i b o s o m a l  RNase a c t e d w i t h o u t a p p r e c i a b l e d e s t r u c t i o n o f endogenous r i b o somal RNA.  Gilbert  (263) found t h a t i n the presence o f p o l y U ,  treatment o f ribosomes w i t h p a n c r e a t i c RNase i n the c o l d had no e f f e c t .  D e l i h a s (30 2) found t h a t w i t h h i s ribosome p r e -  p a r a t i o n o n l y 3% of the t o t a l absorbance  a t 260 nm was  released  i n the presence o f p a n c r e a t i c RNase and t h e r e was no e f f e c t on ribosomal a c t i v i t y .  Ehresmann and E b e l (303) found t h a t T i  RNase caused l e s s d e g r a d a t i o n i n whole 70S ribosomes  than when  the treatment was done on 30S and 50S r i b o s o m a l s u b u n i t s .  At  l e a s t 35-40% o f the 16S RNA was a c c e s s i b l e t o n u c l e a s e a c t i o n . Gupta e t a l . (30 4) found t h a t RNase T i degraded  a l l parts of  the fz RNA except t h a t p r o t e c t e d by the a t t a c h e d ribosomes. Rich's group  (305) o b t a i n e d s i m i l a r r e s u l t s w i t h p a n c r e a t i c  RNase. Equipped w i t h t h i s  i n f o r m a t i o n an e x t e n s i v e c h a r a c t e r i z a -  t i o n o f the low m o l e c u l a r weight RNA bound t o E_^ c o l i was then c a r r i e d o u t .  The r e s u l t s  from T a b l e I I I c l e a r l y  t h a t the tRNA bound t o WRib has a c c e p t o r a c t i v i t y amino a c i d s .  ribosomes show  f o r a l l the  A s i m i l a r phenomenon was observed r e c e n t l y f o r  r a b b i t r e t i c u l o c y t e ribosomes  (276) .  The r e s u l t s o f C u l p e t a l .  135  (276)  d i f f e r e d considerably  from t h o s e shown i n T a b l e I I I .  They a l s o gave r e s u l t s f o r r e t i c u l o c y t e tRNA w h i c h were d i f f e r e n t again  from t h a t bound t o t h e i r r i b o s o m e s .  McNamara (277) have a l s o done a c c e p t o r  S m i t h and  s t u d i e s on r a b b i t  r e t i c u l o c y t e tRNA and t h e i r r e s u l t s d i f f e r from t h o s e o f C u l p e t a l . (276). niques.  T h i s may be due t o d i f f e r e n t i s o l a t i o n  tech-  The r e s u l t s o f S t e a m and H o r o w i t z (280) , u s i n g t h e  t o t a l RNA from N e u r o s p o r a c r a s s a , compare q u i t e  favourably  w i t h many o f t h e s p e c i f i c a c t i v i t i e s shown i n T a b l e I I I . I t s h o u l d be s t r e s s e d t h a t t h e amino a c i d a c c e p t o r were n o t c a r r i e d o u t under optimum c o n d i t i o n s .  studies  For instance,  t h e r a t i o o f Mg /ATP w i l l d e t e r m i n e t h e degree o f a t t a c h m e n t ++  of t h e amino a c i d s t o t h e tRNAs (2 78).  Some s y n t h e t a s e s  r e q u i r e t h e p r e s e n c e o f a s u l f h y d r y l group w h i l e o t h e r s (275,  279).  optimal  do n o t  S i n c e each tRNA s p e c i e s has p r o b a b l y d i f f e r e n t  c o n d i t i o n s f o r maximal amino a c i d c h a r g i n g ,  the present  e x p e r i m e n t s do n o t , t h e r e f o r e , l e a d t o q u a n t i t a t i v e r e s u l t s . Under t h e c o n d i t i o n s u s e d , t h e maximum amino a c i d i n c o r p o r a t i o n f o r each tRNA was o b t a i n e d .  The main p o i n t t h a t  should  be e s t a b l i s h e d h e r e i s t h a t t h e ribosomes c o n t a i n s p e c i e s o f tRNA w h i c h have a c c e p t o r  activity  f o r a l l t h e amino a c i d s and  t h a t t h e amount o f a c c e p t a n c e f o r each amino a c i d v a r i e s .  This  may be due t o t h e r e l a t i v e amounts o f t h e s p e c i f i c tRNA bound t o t h e r i b o s o m e s , o r due t o c o n d i t i o n s under w h i c h t h e a s s a y s were c a r r i e d o u t , o r b o t h .  B a r t z e t a l _ . (291) found t h a t  a r e f l u c t u a t i o n s i n t h e amounts o f s p e c i f i c tRNAs i n d u r i n g c e l l growth.  F o r example, tRNA  there  coli  r e a c h e d i t s maximum  136  l e v e l i n the s t a t i o n a r y phase of c e l l growth whereas tRNA reached i t s maximum l e v e l d u r i n g declined  a f t e r that.  somal p r e p a r a t i o n s  Cvs  the e a r l y l o g phase and  2  then  I t i s also possible that d i f f e r e n t ribo-  w i l l have d i f f e r e n t amounts o f  specific  tRNAs bound o r i t c o u l d be  t h a t c e r t a i n s p e c i f i c tRNAs  more s t r o n g l y bound t o the  ribosomes than o t h e r s .  are  From  the  p r e v i o u s d i s c u s s i o n , i t seems very u n l i k e l y t h a t the v a r i a t i o n i n the  r e l a t i v e amounts o f s p e c i f i c tRNAs bound t o ribosomes  i s due  to the removal o f a p o r t i o n o f some s p e c i f i c tRNAs by  the a c t i o n o f e x o n u c l e a s e s .  The  problem of n u c l e a s e contamin-  a t i o n ; f o r example, f i n g e r n u c l e a s e s , i n v a r i o u s systems has far  o c c u p i e d the  as t h i s l a b o r a t o r y  instance  types of  a t t e n t i o n o f many i n v e s t i g a t o r s .  i s concerned, s e p a r a t i o n s  on B D - c e l l u l o s e  As  of tRNA, f o r  columns a t room temperature f o r up  to  f o u r days have been s u c c e s s f u l l y c a r r i e d out w i t h o u t degradat i o n of the tRNA and w i t h f u l l r e t e n t i o n o f the amino a c i d acceptance.  One  i s presently  t o c a l c u l a t e , from the  impossible  capacity  for  s h o u l d a l s o b e a r i n mind t h a t i t a v a i l a b l e data,  the maximum number of tRNA molecules which c o u l d be bound t o a c t i v e ribosomes because one would not know what percentage o f the  ribosomes' was  actually functional.  i n which the s t u d i e s be pursued i n the The ribosomes  i s another area  on tRNA bound t o ribosomes w i l l have t o  future.  d i s t r i b u t i o n of low (Table  This  IV)  Busch's group (19 0)  m o l e c u l a r weight RNA  p a r a l l e l s exactly  the  from  results obtained  f o r t h e i r f r a c t i o n a t i o n of both low  c u l a r weight n u c l e o l a r  and  r i b o s o m a l RNA  coli  of N o v i k o f f  by  mole-  hepatoma  137 ascites  cells.  A c c o r d i n g t o Tables I I and IV the A eo 2  RNA v a r i e d between 0.58 molecules RNA.  r a t i o o f 4S : 5S  and 0.90 which amounted t o 0.9 t o 1.3  o f tRNA bound t o the ribosomes per molecule  The percentage  o f 5S  of t o t a l RNA bound t o ribosomes i n the  form o f tRNA v a r i e d between 1.3 and 1.7% w h i l e the percentage of the t o t a l RNA bound t o ribosomes i n the form of 5S v a r i e d between 1.5 and 2.8%. these cases  RNA  The l a t t e r v a l u e s i n both of  compare q u i t e f a v o u r a b l y t o those o b t a i n e d by  other i n v e s t i g a t o r s  (55, 286).  S i m i l a r l y , these v a l u e s  q u i t e f a v o u r a b l y t o the expected tRNA u s i n g m o l e c u l a r weights  compare  amounts of rRNA, 5S RNA and  as the only c r i t e r i o n .  I t should  be borne i n mind t h a t some 4.5S RNA was p r e s e n t i n the 5S fractions  ( F i g u r e s 10, 12 and 12a) w h i l e o n l y a s l i g h t  was p r e s e n t i n the 4S RNA  fractions  4-5S RNA RNA  r e g i o n was  fraction  4.5S RNA  and t h i s was  ( F i g u r e s 12 and 12a).  amount  ( F i g u r e s 10 and 25) .  i n s t a n c e , 2.7% of the t o t a l low m o l e c u l a r weight RNA  RNA  For  i n the  a l l found i n the 5S  Thus the amount o f tRNA  bound t o the ribosomes c a l c u l a t e d on the b a s i s o f the amount o f 5S RNA s h o u l d be s l i g h t l y h i g h e r .  The 4.5S RNA  r e p r e s e n t s much l e s s than one molecule per molecule The 4.5S RNA  in  to be a unique  also of 5S  RNA.  c o l i has been shown by the Cambridge group  component and not an a r t e f a c t and work i s w e l l  underway i n sequencing the f u n c t i o n f o r t h i s  this p a r t i c u l a r species  (30 8 ) .  component has not been c l a r i f i e d .  As y e t , The  m o b i l i t y o f t h i s s p e c i e s w i t h r e s p e c t t o 4S and 5S RNA has been v e r i f i e d by Dixon's group (309) .  138 An attempt was made t o c h a r a c t e r i z e f u r t h e r the low molec u l a r weight RNA bound t o i n a 10% p r e p a r a t i v e  c o l i ribosomes by e l e c t r o p h o r e s i s  g e l ( F i g u r e 17) .  The main absorbance  peak d i d n o t correspond t o t h e main peak o f acceptance a c t i v i t y . E f f o r t s were made t o c h a r a c t e r i z e t h i s main absorbance peak. Originally  i t was thought t h a t GTP would be found i n t h i s peak  s i n c e i t i s a major requirement i n p r o t e i n s y n t h e s i s b u t paper chromatography r e v e a l e d indicative  only  two components—one a t the o r i g i n  of a l a r g e m o l e c u l a r w e i g h t s p e c i e s  beyond t h e GTP s t a n d a r d .  and one moving  The component a t t h e o r i g i n gave t h e  t y p i c a l RNA t r a c i n g ( F i g u r e 19).  Further  attempts t o charac-  t e r i z e t h i s peak by chromatography on D E A E - c e l l u l o s e Sephadex A-50 columns r e s u l t e d i n only  o r DEAE-  a s i n g l e peak b e i n g  e l u t e d i n t h e v o i d volume i n each case. At the p r e s e n t  moment the c h a r a c t e r i z a t i o n o f the main  absorbance peak remains t o be e s t a b l i s h e d .  Besides  containing  some tRNA, t h i s p r i n c i p a l peak must c o n t a i n RNA o f lower molec u l a r w e i g h t s i n c e presumably t h i s would be e l u t e d f i r s t the p r e p a r a t i v e  gel.  I t i s q u i t e p o s s i b l e t h a t t h i s RNA i s  a c t u a l l y tRNA t h a t has been p a r t i a l l y degraded. confirm  from  In o r d e r t o  t h i s i t would be necessary t o s e p a r a t e t h i s RHA from  the i n t a c t tRNA b u t u n f o r t u n a t e l y  t h i s cannot be done  A l k a l i n e h y d r o l y s i s s t u d i e s o f t h i s RNA s p e c i e s whether the sample i s tRNA b u t u n f o r t u n a t e l y  should  and the l i m i t e d  mation t h a t c o u l d have been o b t a i n e d  confirm  insufficient  m a t e r i a l was a v a i l a b l e t o c a r r y o u t the a n a l y s e s . the e x p e r i m e n t a l d i f f i c u l t i e s  readily.  Because o f  amount o f i n f o r -  the c h a r a c t e r i z a t i o n o f  139 t h i s peak was  l e f t for future  consideration.  A t t h i s stage i n the t h e s i s the  following additional  p o i n t s had been e s t a b l i s h e d i n a d d i t i o n to those p r e v i o u s l y mentioned  (see p. 132-133): (a) the ribosomes c o n t a i n  of tRNA which have a c c e p t o r  activity  f o r a l l the  species  amino a c i d s  and  t h a t the amount of acceptance f o r each amino a c i d v a r i e d ,  (b)  1-2  molecules o f tRNA are bound t o the  c u l e of 5S RNA, WRib was one  confirmed and  this species  molecule per molecule o f 5S According  was  (c) the presence o f 4.5S  t o my  RNA  bound t o  represented  coli  much l e s s than  RNA.  h y p o t h e s i s on c h a i n - t e r m i n a t i o n ,  thoroughly d i s c u s s e d  e x i s t s i t must be  ribosomes per mole-  which  previously, i f a terminating  t i g h t l y bound t o the  ribosomes and  tRNA probably  i t would occupy a s i t e on the ribosome d i s t i n c t from the mal  tRNA s i t e s .  Such a s p e c i e s  o f RNA  t o exchange w i t h the normal tRNA. ments were c a r r i e d out w i t h two  would not be  Therefore,  expected  exchange e x p e r i -  purposes i n mind: (1) to  i f some tRNAs are more t i g h t l y bound to ribosomes than and  (2) to c o n f i r m  nor-  the presence o r absence of a  see  others,  terminating  tRNA. I t had been shown by  numerous i n v e s t i g a t o r s t h a t exchange  between f r e e tRNA and  tRNA bound t o ribosomes r e a d i l y takes  place  306).  (148, 249,  charging  250,  tRNA w i t h amino a c i d s o r by  w i l l not wash o f f i n high e a s i l y be The  T h i s exchange was  d i s p l a c e d by  Mg  + +  not  temperature.  ion concentration  a f f e c t e d by Bound tRNA  but  could  f r e e tRNA from the s u r r o u n d i n g medium.  n o n s p e c i f i c a s s o c i a t i o n o f tRNA and  ribosomes took p l a c e  in  140 the c o l d and d i d not r e q u i r e the supernatant o r an energy source ing  components.  as by Cannon e t a l . (148). pyrimidine-requiring H - u r a c i l and  was  considered  ribosomal  p e l l e t had  Ribosomes were p r e p a r e d  bound tRNA was  washed i n the u s u a l manner.  almost 3.5 24).  remained v i r t u a l l y c o n s t a n t w i t h i n e x p e r i When l a b e l l e d  removed (Table V I ) .  on a t o t a l r i b o s o m a l  RNA  The  b a s i s had  A 6o 2  about 5.7  increased  be e x p l a i n e d by  1-2  not undergone exchange.  and N o l l  These  (262)  Rich  Cannon  o n l y one b i n d i n g s i t e  ribosome i n the absence of mRNA.  of messenger the amount o f b i n d i n g doubled.  The  per  In the presence Other i n v e s t i g a t o r s  have suggested the presence of t h r e e s i t e s , two  acyl-tRNA and one  f o r peptidy1-tRNA.  s i t e was  only i n the absence of p e p t i d e bond  occupied  (260)  f o r mammalian ribosomes  Q u a n t i t a t i v e s t u d i e s by  e t a l . (14 8) showed t h a t t h e r e was  (285)  (Figure  c o n s i d e r i n g a m u l t i p l i c i t y of tRNA  (see I n t r o d u c t i o n p. 22-24) .  o r 50S  to  molecules o f tRNA  b i n d i n g s i t e s p r e v i o u s l y p o s t u l a t e d by Warner and as w e l l as by W e t t s t e i n  of  molecules of tRNA bound per  as compared w i t h  bound i n a system t h a t has  a l l of  ratio  i n ribosomes exchanged w i t h u n l a b e l l e d tRNA  molecule o f 5S RNA  70S  ribosomal-  exchanged w i t h u n l a b e l l e d tRNA v i r t u a l l y  This represented  r e s u l t s may  Exchange  complete when the number of counts i n the  the l a b e l l e d tRNA was : 5S RNA  from a  c o l i mutant grown i n the presence of  thoroughly t o be  c a r r i e d out i n the same manner  mental e r r o r (Table V, F i g u r e 23).  4S  ATP  o t h e r than the thermal energy of the r e a c t -  An exchange experiment was  3  enzymes, GTP,  second  f o r amino-  aminoacyl-tRNA formation.  141  According  to  p o s s i b i l i t y  of  are  not  the  property of  l a b e l l e d  has  one  the  tRNA  i n  ribosome  t i g h t l y The  a l t e r i n g  consider  two  can  molecules  are  but  bound.  complete  on  A  the  14-24). that  The  other  ments  data  although active  as  s i t e  be  were a f t e r the  sites  tRNA  loosely.  loss  with  and  binding  of  tRNA  after  found the  to  be  i n i t i a l  o r i g i n a l  rapid can  The  place  a  tRNA  the  for  already  here  do  not  sites  d i f f e r e n t exchange  i n a c t i v e i n  the  of  these  preparation.  Many  s i t e tRNA  have  the  two  f i r s t  tRNA  a v a i l a b l e  given  the  (pages  p o s s i b i l i t y  d i f f e r e n t  require-  conditions. (Figure  p r o t e i n  exchange  can  both  sites  been  tRNA  One  f u l l  for  exclude with  with  to  second  would  binding  has  bound  e i t h e r  are  tRNA  ribosome  f i r s t .  structure  binding  f o r  d i s s o c i a t e a  binding;  loose  of  characterized  by  i f both  loss  the  would  number  l a s t  binding  e q u i l i b r i u m  be  the  under  the  f i r s t ,  of  binding  demonstrated  i n  the  but  t i g h t  discussion  the  take  a  the  suggested  s i t e .  Such  weakens  have  are  complex  f o r  they  models  d i s p l a c i n g  t i g h t l y  presented  Unfortunately ribosomes  and  f o r  kinds  could  the  they  the  model  the  e i t h e r  the  f o r  binds  exchange  are,  equivalent one  In  an  eliminate  account  Two  second  not  there  any  tRNA.  and  does  are  might  would  slow  ribosome  to  tRNA  associated  molecules  tRNA  of  bound  bound  exchange  they  exchange  the  i f  The  equivalent  bind  but  tRNA.  the  having  this  i f  This  bound  which  molecule  rapid  binding  s o l u t i o n .  i s  or  exchange  on  slowly.  alone  s i t e s ,  others.  and  s i t e  s i t e  very  that  ribosomal  binding  the  a l _ . (148)  s e v e r a l  the  the  the  et  a l l equivalent,  a b i l i t y  as  Cannon  23)  the  synthesis  ribosomes reasons  were  could  as explain  142 this loss i n a c t i v i t y .  For example, because of the  increased  number o f tRNAs bound to the ribosomes as compared t o 5S a l l the a v a i l a b l e s i t e s on the  ribosome may  the c o n f o r m a t i o n of the ribosome may  be  be b l o c k e d  t h a t 5S RNA by h i g h  protein synthesis.  l e v e l s of tRNA (287).  give no e x p l a n a t i o n was  I t has  c o u l d be r e l e a s e d from the  50S  ribosomal  f o r t h i s phenomenon.  could  T h e i r only  suggestion  t h a t i n c e l l s where most of the ribosomes were i n v o l v e d i n  and  i t c o u l d be d i s p l a c e d by  a t one  previous  of these s i t e s  s t u d i e s on  c u l e s o f 5S RNA  80S  have occupied  (2 88) .  of two  were bound per l a r g e s u b u n i t but  They a l s o found t h a t w i t h the  i n b i o l o g i c a l a c t i v i t y may  tRNA.  have been due  The  only  Their mole-  one  l o s s o f 5S RNA  They suggest t h a t the  the c o n f o r m a t i o n o f the ribosome and to the l o s s of 5S RNA.  sites  i n c l u d e d i n the b i n d i n g mix-  a loss i n b i o l o g i c a l a c t i v i t y .  the 50S  one  ribosomes demonstrated t h a t two  molecule per s u b u n i t when tRNA was  RNA,  there-  subunit  S a r k a r and Comb (288)  may  was  and  may  f a c t t h a t n a t i v e 5S RNA  present  on  r i b o s o m a l s u b u n i t s , when exchanged w i t h i s o l a t e d 5S  on the ribosome was  polypeptide  unique and  to a conformational  5S  essential for biological  and q u i t e d i f f e r e n t from i s o l a t e d 5S RNA.  p o s s i b l e reasons f o r the due  loss  have no r e l a t i o n s h i p  y i e l d e d p a r t i c l e s completely i n a c t i v e i n  activity  there  to s l i g h t changes i n  s y n t h e s i s , suggested t o them t h a t the c o n f o r m a t i o n of the RNA  way  a l s o been found  p r o t e i n s y n t h e s i s , 5S RNA  ture  and/or  a l t e r e d i n such a  as t o prevent the normal exchange from t a k i n g p l a c e fore preventing  RNA,  loss i n b i o l o g i c a l a c t i v i t y change i n a p r o t e i n , a l o c a l  Other could  be  rearrange-  143  merit o f a h e l i c a l p o r t i o n o f t h e RNA o r an a l t e r a t i o n i n some protein-RNA a s s o c i a t i o n .  I n a c t i v a t i o n by a r i b o s o m a l  RNase i s  remote due t o t h e f a c t t h a t t h e exchange took p l a c e a t a h i g h magnesium c o n c e n t r a t i o n and a t 0°; c o n d i t i o n s underwhich t h e ribosomes a r e q u i t e s t a b l e . Although  a f t e r t h e exchange e x p e r i m e n t t h e ribosomes were  found t o be i n a c t i v e and i d e a l l y one w o u l d have hoped f o r t h e maintenance o f a c t i v i t y , t h e u l t i m a t e purpose o f t h e e x p e r i m e n t was t o s t u d y  t h e b i n d i n g a b i l i t y o f s p e c i f i c tRNAs t o t h e s e  ribosomes and t o check f o r t h e p r e s e n c e o f a t e r m i n a t i n g t R N A — b o t h o f w h i c h c o u l d s t i l l be s t u d i e d (see p. 1 3 9 ) . Therefore  a f t e r t h e exchange, t h e r i b o s o m a l - b o u n d tRNA  was f r a c t i o n a t e d on a B D - c e l l u l o s e column t o s e e i f t h e l a b e l l e d tRNA t h a t remained bound t o spread evenly  c o l i ribosomes was  throughout the e l u t i o n p r o f i l e , or whether i t  was c o n c e n t r a t e d  i n a certain region.  The l a b e l l e d tRNA was  spread uniformly throughout the region e l u t e d w i t h the NaCl g r a d i e n t b u t t h e r e was a peak o f r a d i o a c t i v i t y i n t h e e t h a n o l gradient  (Figure 26).  Counts i n t h e peak r e g i o n were 5-10  f o l d h i g h e r than t h a t recorded i n the s a l t g r a d i e n t .  T h i s peak  e t h a n o l f r a c t i o n was a l s o found t o c o n t a i n 4.5S RNA.  Although  the counts i n t h i s e t h a n o l f r a c t i o n were much h i g h e r t h a n  those  r e c o r d e d i n t h e s a l t g r a d i e n t most o f i t c o u l d be a c c o u n t e d f o r i n t h e f o l l o w i n g way: (1) t h e p r e s e n c e o f 4.5S RNA w h i c h w o u l d be l a b e l l e d .  T h i s 4.5S RNA, w h i c h w o u l d t e n d t o remain s t r o n g l y  bound t o t h e B D - c e l l u l o s e column, w o u l d be e l u t e d i n h i g h e t h a n o l c o n c e n t r a t i o n s w h i c h w o u l d overcome t h e h y d r o p h o b i c  144 i n t e r a c t i o n s between the RNA  and the r e s i n ,  (2) s i n c e the  e t h a n o l f r a c t i o n has been found t o c o n t a i n a l l S e r , T y r Trp a c c e p t o r a c t i v i t i e s , about  17% of the Leu a c c e p t o r  and 9% of the Phe a c c e p t o r a c t i v i t y would be expected presence was  i n this  (295) , t h i s  to g i v e h i g h e r counts due fraction  and activity  r e g i o n alone  t o t h e i r combined  (see T a b l e I I I ) , (3) s i n c e t R N A  found t o be p r e s e n t i n the l a r g e s t amount on  ribosomes,  presumably i t alone c o u l d account f o r some of the  counts  (Table I I I ) ; however, t h e r e i s no reason t o b e l i e v e t h a t p a r t i c u l a r tRNA i s l e s s v u l n e r a b l e t o exchange than The reasons  a d d i t i o n a l experiments  T r p  one  another.  t o c o n f i r m these r e s u l t s  c o u l d not be c a r r i e d out were: (a) a c c e p t o r s t u d i e s w i t h Trts tRNA one  - g i v e s a very h i g h background i s d e a l i n g w i t h such  low counts  (up to 300 (250 cpm),  i m p o s s i b l e t o get a d e f i n i t i v e r e s u l t , experiments  cpm)  and s i n c e  i t would be  (b) d o u b l e - l a b e l l i n g  c o u l d not be c a r r i e d out because the background  l e v e l s would be h i g h e r than the counts t h a t were b e i n g i n v e s t i gated h e r e ,  (c) the assay used t o determine  acceptance  activity  of s p e c i f i c tRNAs would i n v o l v e the p r e c i p i t a t i o n of a l l the tRNAs i n the e t h a n o l f r a c t i o n . I t may  be p o s s i b l e t o s e p a r a t e the 5 a c c e p t o r a c t i v i t i e s  found i n the e t h a n o l f r a c t i o n on a Kelmer  reversed-phase  column b u t , once a g a i n , s i n c e one i s d e a l i n g w i t h such counts  any s m a l l e r r o r , f o r example, i n background  would l e a d t o an o v e r a l l l a r g e e x p e r i m e n t a l e r r o r . experiments and i t was  low  counts, These  would have been very complex and time consuming f e l t t h a t any  r e s u l t s o b t a i n e d would not have added  145 significantly  t o the c o n c l u s i o n s a l r e a d y e s t a b l i s h e d .  T a k i n g a l l these f a c t o r s i n t o c o n s i d e r a t i o n , t h e r e would be very few counts l e f t over which  c o u l d be a l l o c a t e d t o the  presence o f a s p e c i f i c c h a i n - t e r m i n a t i n g tRNA.  This species  o f tRNA would not only be l a b e l l e d but would a l s o be e x p e c t e d to be p r e s e n t as a sharp peak o f r a d i o a c t i v i t y . 50,000 counts a t t r i b u t e d mainly t o tRNA was the B D - c e l l u l o s e column  Approximately  distributed  ( F i g u r e 24, T a b l e V I ) .  over  I f a mere 1%  of t h i s tRNA c o u l d be c o n s i d e r e d due t o the presence o f a s p e c i f i c tRNA t e r m i n a t o r , then t h e r e would be enough r a d i o a c t i v i t y p r e s e n t such t h a t i t would show up as a d i s t i n c t sharp peak.  C a l c u l a t i n g on the b a s i s o f 5S RNA,  the counts  remaining would i n d i c a t e l e s s than one molecule o f 4S m a t e r i a l f o r every 20 5S RNA  molecules.  Thus the presence o f a  s p e c i f i c t i g h t l y bound t e r m i n a t i n g tRNA i s not p o s s i b l e . i In summary, tRNA exchange r e s u l t e d i n i n c r e a s e d b i n d i n g o f tRNA t o the ribosomes; from 1-2 6 molecules per molecule o f 5S RNA. c o n d i t i o n s used, was  almost 100%  of ribosomal a c t i v i t y .  molecules t o approximately Exchange, under  the  and a l s o r e s u l t e d i n the l o s s  The tRNA which  d i d not exchange  was  s p r e a d u n i f o r m l y throughout the e l u t i o n p a t t e r n s u g g e s t i n g t h a t a l l tRNAs are p r o b a b l y bound t o the ribosome degree, t h a t i s , other.  t o the same  one tRNA i s not bound more s t r o n g l y than an-  The absence o f a sharp peak o f r a d i o a c t i v i t y i s  f u r t h e r p r o o f o f the absence o f a s p e c i f i c c h a i n - t e r m i n a t i n g tRNA, s i n c e as p r e v i o u s l y d i s c u s s e d , such a s p e c i e s o f tRNA would be expected t o be not only t i g h t l y bound to the r i b o somes b u t a l s o non-exchangeable  w i t h tRNA.  146  BIBLIOGRAPHY 1.  Zamecnik, P . C , C o l d S p r i n g Harbor 34, 1 ( 1 9 6 9 ) .  Sym. Q u a n t .  2.  Tissieres,  3.  B o l t o n , E . T . , H o y e r , W.H., a n d R i t t e r , D.B., i n R.B. R o b e r t s ( E d i t o r ) , M i c r o s o m a l P a r t i c l e s and P r o t e i n S y n t h e s i s , Pergamon P r e s s , New Y o r k , 1958, p . 18.  4.  R o b e r t s , R.B. ( E d i t o r ) ,  ibid.  5.  R o b e r t s , R.B., B r i t t e n , p . 84.  R.J.,  6.  T i s s i e r e s , A., W a t s o n , J.D., S c h l e s s i n g e r , D., a n d H o l l i n g w o r t h , B.R., J . M o l . B i o l . , 1, 221 ( 1 9 5 9 ) .  7.  K u r l a n d , C.G., J . M o l .  8.  G o r i n i , L., and K a t a j a , E . , P r o c . N a t . Acad. 51, 487 ( 1 9 6 4 ) .  9.  G u t h r i e , C., a n d Nomura, M., N a t u r e ,  A., a n d W a t s o n , J.D., N a t u r e ,  Biol.,  Biol.,  182, 778 ( 1 9 5 8 ) .  and B o l t o n , E.T., i b i d . ,  2, 83  (1960). S c i . U.S.A.,  219, 232 ( 1 9 6 8 ) .  10.  H i l l e , M.B., M i l l e r , M.J., I w a s a k i , K., a n d Wahba, A . J . , P r o c . N a t . A c a d . S c i . U.S.A., 5_8, 1652 ( 1 9 6 7 ) .  11.  Nomura, M., a n d L o w r y , C V . , P r o c . N a t . A c a d . 58, 946 ( 1 9 6 7 ) . -  12.  E i s e n s t a d t , J.M. , Brawerman, G., B i o c h e m i s t r y , 5_, 2777 (1966).  13.  R e v e l , M., a n d G r o s , F . , B i o c h e m . B i o p h y s . R e s . Commun., 25, 124 ( 1 9 6 6 ) .  14.  S t a n l e y , W.M., J r . , S a l a s , M., Wahba, A . J . , a n d O c h o a , S., P r o c . N a t . A c a d . S c i . U.S.A., 5_6 , 290 ( 1 9 6 6 ) .  15.  R e v e l , M. , L e l o n g , J . C , Brawerman, G. , a n d G r o s , F. , N a t u r e , 219_, 1016 ( 1 9 6 8 ) .  16.  Wahba, A . J . , C h a e , Y.B., I w a s a k i , K., Mazumder, R., M i l l e r , M . J . , S a b o l , S., a n d S i l l e r o , M.A.G., C o l d S p r i n g H a r b o r Sym. Q u a n t . B i o l . , 3_4, 285 ( 1 9 6 9 ) .  17.  Clark, B.F.C, (1966) .  18.  Steitz,  and Marcker,  J.A., Nature,  K.A., J . M o l .  224, 957 ( 1 9 6 9 ) .  S c i . U.S.A.,  Biol.,  17, 394  147  19.  H i n d l e y , J . , and  20.  H e r z b e r g , M. , L e l o n g , J . C , _4_4, 297 (1969).  21.  Greenshpan,  22.  L u c a s - L e n a r d , J . , and L i p m a n n , F., U.S.A., 57, 1050 (1967).  23.  G h o s h , H.P., and K h o r a n a , U.S.A., 58, 2455 ( 1 9 6 7 ) .  24.  Nomura, M. , Lowry, C.V., and G u t h r i e , C , A c a d . S c i . U.S.A., 5_8, 1487 (1967).  25.  K o l a k o f s k y , D., 244 (1968).  26.  E r t e l , R., Weissbach, (1968).  27.  Ono, Y., S k o u l t c h i , A., K l e i n , A., N a t u r e , 220, 1304 (1968).  28.  Ono, Y., S k o u l t c h i , A., W a t e r s o n , J . , and N a t u r e , 222, 645 (1969).  29.  G r u n b e r g - M a n a g o , M., C l a r k , B.F.C., R e v e l , M., Rudland, P.S., and Dondon, J . , J . M o l . B i o l . , 40_, 33 ( 1 9 6 9 ) .  30.  M o n r o , R.E.,  31.  L u c a s - L e n a r d , J . , and H a e n n i , U.S.A. , 6_3, 93 (1969) .  32.  E r b e , R.H., Nau, 441 ( 1 9 6 9 ) .  33.  C r a v e n , G.R., Voynow, P., H a r d y , S . J . S . , and G. , B i o c h e m i s t r y , 8, 2906 ( 1 9 6 9 ) .  34.  T r a u t , R.R., D e l u i s , H. , Ahmad-Zadeh, C , B i c k l e , T.A. , P e a r s o n , P., and T i s s i e r e s , A., C o l d S p r i n g H a r b o r Sym. Q u a n t . B i o l . , 34, 25 ( 1 9 6 9 ) .  35.  M o o r e , P.D., T r a u t , R.R., N o l l e r , H., P e a r s o n , D e l i u s , H., J . M o l . B i o l . , 31, 441 ( 1 9 6 8 ) .  36.  K u r l a n d , C.G., Voynow, P., H a r d y , S . J . S . , R a n d a l l , L . , and L u t t e r , L . , C o l d S p r i n g H a r b o r Sym. Q u a n t . B i o l . , 34, 17 ( 1 9 6 9 ) .  H.,  S t a p l e s , D.H.,  and  and  R e v e l , M.,  Ohta,  T.,  Nature,  H.G.,  and  2_2_4, 964  (1969).  R e v e l , M. , J . M o l .  Nature,  224,  331  P r o c . Nat.  P r o c . Nat.  Thach,  Biol.,  (1969).  Acad. S c i .  Acad. S c i . Proc.  R.E.,  Nat.  Nature,  220,  B r o t , N., R e d f i e l d , B., A l l e n d e , J . E . , and H. , P r o c . N a t . A c a d . S c i . U.S.A., 5_9, 861  J . Mol.  M.,  Biol.,  and  and  26_, 147 A.,  Lengyel,  Lengyel,  P.,  (1967).  P r o c . Nat.  L e d e r , P.,  P.,  J . Mol.  Acad. S c i . Biol.,  39,  Kurland,  P.,  C.  and  148 37.  Sypherd, P.S., O ' N e i l l , D.M., and T a y l o r , M.M., C o l d S p r i n g Harbor Sym. Quant. B i o l . , 3_4, 77 (1969) .  38.  Mizushima,  39.  Hershey, J.W.B., Dewey, K.F., and Thach, R.E., Nature, 222, 944 (1969).  40.  P a r e n t i - R o s i n a , R., E i s e n s t a d t , A., and E i s e n s t a d t , J.M., Nature, 221, 363 (1969).  41.  Kaempfer, R.O.R., Meselson, M., and Raskas, H.J., J . Mol. B i o l . , 31, 277 (1968).  42.  Brownlee, G.G., Sanger, F., and B a r r e l l , 215, 735 (1967).  43.  K l e i n , H.A., and C a p e c c h i , M.R., J . B i o l . Chem., 246, 1055 (1971).  44.  S t a n l e y , W.M., 1302 (1965).  45.  M i d g l e y , J.E.M., Biochem. Biophys. A c t a , 10 8, 348 (1965).  46.  M c l l r e a v y , D.J., and M i d g l e y , J.E.M., Biochem. Biophys. A c t a , 142, 47 (1967).  47.  F e l l n e r , P., and Sanger, F., Nature, 219, 236 (1968).  48.  Smith, I . , Dubnau, D., M o r e l l , P., and Marmur, J . , J . Mol. B i o l . , 33, 123 (1968).  49.  Aronson, A . I . , J . M o l . B i o l . ,  50.  Takanami, M., J . M o l . B i o l . ,  51.  M o l l e r , W., and Boedtker, H., E d i t i o n s du Centre N a t i o n a l de l a Recherche S c i e r i t i f i q u e , P a r i s , 99 (1962).  52.  L e p p l a , S.H., Ph.D. T h e s i s , U n i v e r s i t y o f W i s c o n s i n , Madison, W i s c o n s i n (1969).  53.  Woese, C.R., Nature, 220, 923 (1968).  54.  A t t a r d i , G., Huang, P.C., and Kabat, S., Proc. Nat. Acad. S c i . U.S.A., 5_3, 1490 (1965).  55.  Rosset, R., Monier, R., and J u l i e n , J . , B u l l . B i o l . , 46, 87 (1964).  56.  Z e h a v i - W i l l n e r , T., and Comb, D.G., J . M o l . B i o l . , 16, 250 (196.6).  S., arid Nomura, M., Nature, 226, 1214 (1970).  B.G., Nature,  J r . , and Bock, R.M., B i o c h e m i s t r y , £ ,  5, 453 (1962). 23, 135 (1967).  Soc. Chim.  149 57.  Young, R . J . , B i o c h e m i s t r y ,  58.  Fellner,  59.  D o t y , P., B o e d t k e r , H., F r e s c o , J.R., H a s e l k o r n , L i t t , M. , P r o c . N a t . A c a d . S c i . U.S.A., 45^, 482  60.  Traub,  P., and Nomura, M. , J . M o l . B i o l . ,  3_4, 5 7 5 ( 1 9 6 8 ) .  61.  Traub,  P., a n d Nomura, M. , J . M o l . B i o l . ,  40_, 3 9 1 ( 1 9 6 9 ) .  62.  F u r a n o , A.V., B r a d l e y , D.F., a n d C h i l d e r s , L.G., B i o c h e m i s t r y , 5 , 3044 ( 1 9 6 6 ) .  63.  S a n t e r , M. , a n d S m i t h ,  64.  26  7, 2263  (1968).  P., E h r e s m a n n , C., and E b e l , J . P . , N a t u r e , 2 2 5 ,  (1970).  (1966).  Geiduschek,  R., a n d  (1959).  J.R. , J . B a c t e r i o l o g y , 9_2,  P., a n d H a s e l k o r n ,  _38, 6 4 7 ( 1 9 6 9 ) .  1099  R., A n n . Rev. B i o c h e m . ,  65.  Attardi,  66.  M a t t h a e i , H., S a n d e r , G., Swan, O., K r e u z e r , T., C a f f i e r , H. , a n d P a r m e g g i a n i , A., N a t u r w i s s e n s c h a f t e n , 5_5, 2 8 1  G., A n n . Rev. M i c r o b i o l o g y , 21,  383 ( 1 9 6 7 ) .  (1968).  67.  O c h o a , S., N a t u r w i s s e n s c h a f t e n ,  5_5, 5 0 5 ( 1 9 6 8 ) .  68.  C o l d S p r i n g Harbor  69.  Ron, E . Z . , K o h l e r , R.E., a n d D a v i s , B.D., J . M o l . B i o l . ,  70.  S p i r i n , A.S., a n d G a v r i l o v a , L . P . , i n The R i b o s o m e , S p r i n g e r - V e r l a g New Y o r k , I n c . , New Y o r k , 19 6 9 .  71.  Marcker,  72.  C l a r k , B.F.C., a n d M a r c k e r ,  73.  Adams, J.M., and C a p e c c h i , M., U.S.A. , 5_5, 1 4 7 ( 1 9 6 6 ) .  74.  W e b s t e r , R., E n g e l h a r d t , D., and Z i n d e r , N., P r o c . N a t . A c a d . S c i . U.S.A., 5_5, 1 5 5 ( 1 9 6 6 ) .  75.  C a p e c c h i , M. , P r o c . N a t . A c a d . S c i . U.S.A., 5_5,  76.  Smith,  Sym. Q u a n t . B i o l . ,  31, (1966).  36, 83 ( 1 9 6 8 ) .  (1964).  K.A. , a n d S a n g e r , F . , J . M o l . B i o l . , 8, 8 3 5 K.A., N a t u r e ,  2 1 1 , 378 ( 1 9 6 6 ) .  Proc. Nat. Acad.  (1966) . (1968).  A.E., and Marcker,  K.A., J . M o l . B i o l . ,  Sci.  1517  38,241  150 77.  G a l p e r , J . B . , and D a r n e l l , J . E . , Biochem. B i o p h y s . Res. Commun., 34, 205 (1968).  78.  S c h w a r t z , J.H., Meyer, R., E i s e n s t a d t , J.M., and Brawerman, G., J . M o l . B i o l . , 25, 571 (1967).  79.  Bachmayer, H., and K r e i l , G., B i o c h i m . B i o p h y s . A c t a , 169, 95 (1968).  80.  B r u t o n , C . J . , and H a r t l e y , B.S., Biochem. J . , 108, 281 (1968).  81.  D i c k e r m a n , H.W., S t e e r s , E., J r . , R e d f i e l d , B.G., and W e i s s b a c h , H. , J . B i o l . Chem., 242, 1522 (1967).  82.  K o l a k o f s k y , D., Dewey, K.F., Hershey, J.W.B., and Thach, R.E., P r o c . N a t . Acad. S c i . U.S.A., 6 1 , 1066 (1968).  83.  N i s h i z u k a , Y., and Lipmann, F., A r c h . Biochem. B i o p h y s . , 116, 344 (1966).  84.  B r e t s c h e r , M.S., J . M o l . B i o l . , 3£, 131 (1968).  85.  Z i n d e r , N.D., E n g e l h a r d t , D.L., and Webster, R.E., C o l d S p r i n g Harbor Sym. Quant. B i o l . , 3_1, 251 (1966) .  86.  F o x , J . L . , and Ganoza, M.C., Biochem. Commun., 32, 1064 (1968).  87.  S o l i , D., J . M o l . B i o l . , 34, 175 (1968).  88.  Caskey, C.T., Tompkins, R., S c o l n i c k , E., C a r y k , T., and N i r e n b e r g , M., S c i e n c e , 162, 135 (1968).  89.  S c o l n i c k , E., Tompkins, R., Caskey, T., and N i r e n b e r g , M., P r o c . N a t . Acad. S c i . U.S.A., 6 1 , 765 (1968).  90.  M i l m a n , G., G o l d s t e i n , J . , S c o l n i c k , E., and C a s k e y , T., P r o c . N a t . Acad. S c i . U.S.A., 63_, 183 (1969).  91.  S c o l n i c k , E.M., and Caskey, C.T., P r o c . N a t . Acad. S c i . U.S.A., 6±, 1235 (1969).  92.  G o l d s t e i n , J . , M i l m a n , G., S c o l n i c k , E., and C a s k e y , T., P r o c . N a t . Acad. S c i . U.S.A., € 5 , 430 (1970).  93.  Tompkins, R.K., S c o l n i c k , E.M., and Caskey, C.T., P r o c . Nat. Acad. S c i . U.S.A., £ 5 , 702 (1970).  94.  C a s k e y , T., S c o l n i c k , E., Tompkins, R. , G o l d s t e i n , J . , and M i l m a n , G., C o l d S p r i n g Harbor Sym. Quant. B i o l . , 34, 479 (1969).  B i o p h y s . Res.  151  95.  Smrt, J . , Kemper, W., C a s k e y , T., and N i r e n b e r g , M., J . B i o l . Chem., 245, 2753 (1970).  96.  Beaudet, A.L., and Caskey, C T . , N a t u r e , 227, 38 (1970).  97.  G o l d s t e i n , J . L . , Beaudet, A.L., and C a s k e y , C T . , P r o c . Nat. Acad. S c i . U.S.A., £ 7 , 99 (1970).  98.  G o l d s t e i n , J . L . , and Caskey, C T . , P r o c . N a t . Acad. S c i . U.S.A., 67, 537 (1970).  99.  S c o l n i c k , E., M i l m a n , G., Rosman, M., and Caskey, T., N a t u r e , 225, 152 (1970).  100.  Ganoza, M . C , and Tomkins, J.K.N., Biochem. B i o p h y s . Res. Commun., 40, 1455 (1970).  101.  V o g e l , Z., Z a m i r , A., and E l s o n , D. , B i o c h e m i s t r y , j3, 5161 (1969).  102.  C u z i n , F., Ksetchmer, N., G r e e n b e r g , R.E., H u r w i t z , R., and C h a p e v i l l e , F., P r o c . N a t . Acad. S c i . U.S.A., 58, 2079 (1967).  103.  d e G r o o t , N., P a n e t , A., and L a p i d o t , Y., Biochem. B i o p h y s . Res. Commun., 3 1 , 37 (1968).  104.  V o g e l , Z., Z a m i r , A., and E l s o n , D., P r o c . N a t . A c a d . S c i . U.S.A., 61, 701 (1968).  105.  K o s s e l , M., and R a j Bhandary, U.L., J . M o l . B i o l . , 35, 539 (1968).  106.  M e n n i n g e r , J.R., M u l h o l l a n d , M . C , and S t i r e w a l t , Biochem. B i o p h y s . A c t a , 217, 496 (1970).  107.  C a p e c c h i , M.R., and K l e i n , H.A., N a t u r e , 226, 1029 (1970).  108.  N i c h o l s , J . L . , N a t u r e , 225, 147 (1970).  109.  I s h i t s u k a , H., and K a j i , A., P r o c . N a t . Acad. S c i . U.S.A., 66, 168 (1970).  110.  Subramanian, A.R., Ron, E.Z., and D a v i s , B.D., P r o c . N a t . Acad. S c i . U.S.A., 6 1 , 761 (1968).  111.  Subramanian, A.R., and D a v i s , B.D., N a t u r e , 228, 1273 (1970).  112.  S a b o l , S., S i l l e r o , M.A.G., I w a s a k i , K., and Ochoa, S., N a t u r e , 228, 1269 (1970).  W.S.,  152 113.  Subramanian, A.R., D a v i s , B.D., and B e l l e r , R . J . , C o l d S p r i n g Harbor Sym. Quant. B i o l . , 34_, 223 (1969).  114.  A l g r a n a t i , I.D., G o n z a l e z , N.S., and Bade, E.G., Nat. Acad. S c i . U.S.A./ 62, 574 (1969).  115.  S c h l e s s i n g e r , D., M a n g i a r o t t i , G., and A p i r i o n , D., Nat. Acad. S c i . U.S.A., 5 8 , 1782 (1967).  116.  F r i e d m a n , H., L u , P., and R i c h , A., N a t u r e , 223, 909 (1969).  117.  I w a s a k i , K., S a b o l , S., Wahba, A . J . , and Ochoa, S., A r c h . Biochem. B i o p h y s . , 125, 542 (1968).  118.  Chae, Y.B., Mazumder, R., and Ochoa, S., P r o c . Nat. Acad. S c i . U.S.A., 63_, 828 (1969).  119.  Kan, Y.W., G o l i n i , F., and Thach, R.E., S c i . U.S.A., 67, 1137 (1970).  120.  Kaempfer, R., N a t u r e , 228, 534  121.  Takata, R., Tamaki, M.,  122.  Weisblum, B.,  123.  K a l t s c h m i d t , E., and Wittman, H.G., S c i . U.S.A., 6_7, 1276 (1970).  124.  Osawa, S.,  Takata, R.,  125.  O z a k i , M.,  Mizukshima, S.,  126.  S l o b i n , L . I . , Biochem. Biophys.  127.  S t a n l e y , W.M.  128.  K i r t i k a r , D.M.W., and K a j i , A.,  129.  F o r g e t , B.G.,  130.  Jordan, B.R., Feunteun, J . , and Monier, R., B i o l . , 5£, 605 (1970).  493  (1968).  (1969).  (1965).  and Davies, J . , B a c t e r i o l . Reviews, 32,  and Dekio,  Proc. Nat. Acad. S., Molec. Gen.  and Nomura, M., Res.  Genetics,  Nature,  and Weissman, S.M.,  221,  Commun., 3_9_, 470  , J r . , and Bock, R.M. , B i o c h e m i s t r y ,  (1968) .  (1967).  P r o c . Nat. Acad.  (1970).  (1970).  1302  Proc.  Osawa, S., Tanaka, K., Teraoka, H., and Molec. Gen. G e n e t i c s , 109, 123 (1970).  107, 32 (1970) . 333  Proc.  4_,  J . B i o l . Chem., 243, 5345 S c i e n c e , 158,  1695  J . Mol.  153 131.  S i d d i q u i , M.A.Q., and Hosokawa, K., Biochem. B i o p h y s . Res. Commun., 36, 711 (1969).  132.  Raacke, I.D., Biochem. B i o p h y s . Res. Commun., 3 1 , 528 (1968).  133.  C a n t o r , C.R., N a t u r e , 216, 513 (1967).  134.  B o e d t k e r , H., and K e l l i n g , D.G., Biochem. B i o p h y s . Res. Commun., 29-, 758 (1967). V  135.  L e w i s , J.B., and D o t y , P., N a t u r e , 225, 510 (1970).  136.  W i l l i a m s o n , R., and B r o w n l e e , G.G., Fed. E u r . Biochem. Soc. L e t t . , 3, 306 (1969).  137.  S p a d a r i , S. , and R i t o s s a , F., J . M o l . B i o l . , 5_3, 357 (1970).  138.  M i l l e r , O.L., H a n k a l o , B.A., and Thomas, C.A., J r . , S c i e n c e , 169, 392 (1970).  139.  Purdom, I . , B i s h o p , J.O., and B u n s t i e l , M.L., N a t u r e , 227, 239 (1970).  140.  Muto, M., B i o c h e m i s t r y , 9, 3683  141.  J a r r y , B., and R o s s e t , R., Biochem. B i o p h y s . Res. Commun., 41, 789 (1970).  142.  Ehresmann, C., F e l l n e r , P., and E b e l , J.B., N a t u r e , 227. 1321 (1970).  143.  Hartman, K.A., and Thomas, G.J., J r . , S c i e n c e , 170, 740 (1970).  144.  Nomura, M., and Erdmann, V.A., N a t u r e , 228, 744 (1970).  145.  G u t h r i e , C., Nashimoto, H., and Nomura, M., C o l d S p r i n g Harbor Sym. Quant. B i o l . , 3_4, 69 (1969).  146.  N a s h i m o t o , H., and Nomura, H., P r o c . N a t . Acad. S c i . U.S.A., 67, 1440 (1970).  147.  Takanami, M., J . M o l . B i o l . , 23 135 (1967).  148.  Cannon, M. , K r u g , R. , and G i l b e r t , W. , J . M o l . B i o l . , 1_, 360 (1963).  149.  K a j i , A., and K a j i , H., Biochem. B i o p h y s . Res. Commun., 1 3 , 186 (1963).  (1970).  154 150.  K a j i , H., and K a j i , A., P r o c . N a t . Acad. S c i . U.S.A., . 52, 1541 (1964).  151.  S p y r i d e s , G.J., P r o c . N a t . Acad. S c i . U.S.A., 5 1 , 1220 (1964).  152.  P e s t k a , S. , and N i r e n b e r g , M.W. , J . M o l . B i o l . ,''21, 145 (1966).  153.  K u r l a n d , C.G., J . M o l . B i o l . , 18, 90 (1966).  154.  C r i c k , F.H.C., Sym. Soc. Exp. B i o l . , 12, 138 (1968).  155.  C h a p e v i l l e , F., Lipmann, F., von E h r e n s t e i n , G., Weisblum, B., Ray, W.D., J r . , and B e n z e r , S., P r o c . N a t . Acad. S c i . U.S.A., £ 8 , 1086 (1962).  156.  Suzuka, I . , K a j i , H., and K a j i , A., P r o c . N a t . Acad. S c i . U.S.A., 55, 1483 (1966).  157.  M a t t h a e i , H., and M i l b e r g , M., Biochem. Commun., 29_, 593 (1967).  158.  Vazquez, D., and Monro, D.E., B i o c h i m . B i o p h y s . A c t a , 142, 155 (1967).  159.  P e s t k a , S., and N i r e n b e r g , M. , J . M o l . B i o l . , 21, 145 (1966).  160.  Monro, R.E., J . M o l . B i o l . , 26, 147 (1967).  161.  P e s t k a , S., J . B i o l . Chem., 242, 4939  162.  P e s t k a , S., J . B i o l . Chem., 243, 4038 (1968).  163.  K u r i k i , Y., Fukuma, I . , and K a j i , A., J . B i o l . Chem., 244, 1365 (1969).  164.  M c L a u g h l i n , C.S., Dondon, J . , Grunberg-Manago, M., M i c h e l s o n , A.M., and Saunders, G., J . M o l . B i o l . , 32, 521 (1968).  165.  Monro, R.E., C e r n a , J . , and M a r c k e r , K.A., P r o c . N a t . Acad. S c i . U.S.A., 6 1 , 1042 (1968).  166.  K a j i , H., Suzuka, 219 (1966).  167.  Moore, P.B., J . M o l . B i o l . , 18, 8 (1966).  168.  F u r a n o , A.V., B i o c h i m . B i o p h y s . A c t a , 161, 255 (1968).  BiophysRes.  (1967).  I . , and K a j i , A., J . M o l . B i o l . , 18,  155  169.  T r a u t , R.R. , and Haenni, A.L. , Europ. J . Biochem.. 2_, 64 ( 1 9 6 7 ) .  170.  K n i g h t , E., J r . , and Sugiyama, T., P r o c . Nat. Acad. S c i . U.S.A. , 6_3, 1 3 8 3 (1969) .  171.  K n i g h t , E. , J r . , B i o c h e m i s t r y , 8_, 5089  172.  Nass, M.M.K., and Buck, C A . , Proc. Nat. Acad. S c i . U.S.A., 6 2 , 506 ( 1 9 6 9 ) .  173.  B a r n e t t , W.E., and Brown, D.H., Proc. Nat. Acad. S c i . U.S.A., 5 7 , 452 ( 1 9 6 7 ) .  174.  Nass, M.M.K., and Buck, C A . , J . M o l . B i o l . , (1970).  175.  Pere, J . J . , K n i g h t , E., J r . , and D a r n e l l , J.E., J . M o l . B i o l . , 3_3, 609 (1968) .  176.  Ohe, K., Weissman, S.M., and Cooke, N.R., J . B i o l . Chem., 244, 5320 ( 1 9 6 9 ) .  177.  F o r g e t , B.G., and Weissman, S.M., Nature,  178.  S i r b a s k u , D.A., and Buchanan, J.M., J . B i o l . Chem., 2 4 5 , 2693 ( 1 9 7 0 ) .  179.  Bishop, J.M., L e v i n s o n , W.E., Q u i n t r e l l , N., S u l l i v a n , D. , F a n s h i e r , L. , and Jackson, J . , V i r o l o g y , 42!, 182 (1970).  180.  Bishop, J.M., L e v i n s o n , W.E., S u l l i v a n , D., F a n s h i e r , L., Q u i n t r e l l , W. , and Jackson, J . , V i r o l o g y , 42_, 927 ( 1 9 7 0 ) .  181.  Weinberg, R.A. , and Penman, S., J . M o l . B i o l . , (1968).  182.  Weinberg, R.A., and Penman, S., Biochim. Biophys. A c t a , 1 9 0 , 10 ( 1 9 6 9 ) .  183.  Bernhardt, D., and D a r n e l l , A.E., J r . , J . M o l . B i o l . , 4 2 , 43 ( 1 9 6 9 ) .  184.  Mowshowitz, D.B. , J . Mol. B i o l . ,  185.  Gardner, J.A.A., and Hoagland, M.B., J . M o l . B i o l . , 2 4 3 , 10 ( 1 9 6 8 ) .  186.  K i n g , H.W.S., and F i t s c h e n , W., Biochim. Biophys. A c t a , 1 5 5 , 32 ( 1 9 6 8 ) .  (1969).  5_4, 187  2 1 3 , 878  (1967)  3_8, 289  50_, 143 ( 1 9 7 0 ) .  156 187.  Z a p i s e k , W.F.,  istry, 188.  S a p o n a r a , A.G., a n d E n g e r , M.D.,  Biochem-  8, 1170 (1968) .  M u r a m a t s u , M., H o d n e t t , J . L . , a n d B u s c h , H., J . B i o l .  Chem., 241, 1544 (1966). 189.  Nakamura, T., Rapp, F . , a n d B u s c h , H., C a n c e r R e s . , 27,  190.  Nakamura, T., P r e s t a y k o , A.W.,  1084  (1967).  a n d B u s c h , H., J . B i o l .  Chem., 243, 1368 (1968). 191.  P e a c o c k , A . C , a n d Dingman, C W . , B i o c h e m i s t r y ,  6_, 1818  (1967) . 192.  H o d n e t t , J . L . , a n d B u s c h , H., J . B i o l .  Chem.,  243, 6334  (1968) . 193. 19 4.  P r e s t a y k o , A.W.,  47, 505 (1970).  R o - C h o i , T.S., M o r i y a m a , Y., C h o i , Y . C , a n d B u s c h ,  J. 195.  Biol.  209, 161 (1970).  El-Khatib,  S.M. , R o - C h o i , T.S., C h o i , Y . C , a n d B u s c h ,  H., J . B i o l . 197. 198.  M.,  Chem., 245, 1970 (1970).  M o r i y a m a , Y., l p , P., a n d B u s c h , H., B i o c h i m . B i o p h y s .  Acta, 196.  T o n a t o , M., a n d B u s c h , H., J . M o l . B i o l . ,  Chem., 245, 3416 (1970).  B u s c h , H., i n H. B u s c h a n d K. Smetana ( E d i t o r s ) , T h e N u c l e o l u s , A c a d e m i c P r e s s , New Y o r k , 19 70, p . 285. P r e s t a y k o , A.W.,  H., J . B i o l .  T o n a t o , M., L e w i s , B.C., a n d B u s c h ,  Chem., 246, 182 (1971).  199.  S y , J . a n d M c C a r t y , K.S., B i o c h i m . B i o p h y s . A c t a , 228,  200.  Udem, S.A., Kaufman, K., a n d W a r n e r , J.R., J . B a c t e r i o l o g y ,  201.  L i z a r d i , P.M.,  202.  Brownlee,  203.  Hindley, J . , J . Mol. B i o l . ,  204.  Goldstein,  517  (1971).  105, 101 (1971) . 140  (1971).  (1969).  a n d L u c k , D . J . L . , N a t u r e New B i o l o g y , 229,  G.G., N a t u r e New B i o l o g y ,  229, 147 (1971).  30_, 125 (1967).  J . , a n d Harewood, K. , J . M o l . B i o l . ,  3_9, 383  157 205.  A l t m a n , S., N a t u r e New  B i o l o g y , 229, 19  (1971).  206.  I w a b u c h i , M., M i z u k a m i , Y., and Sameshima, M., B i o p h y s . A c t a , 228, 693 (1971).  207.  L a y c o c k , D.G.,  208.  L a b r i e , F., N a t u r e , 221, 1217  (1969).  209.  Dubuy, B., and Weissman, S.M., (1971).  J . B i o l . Chem., 246,  210.  Voynow, P., (1971).  B i o c h e m i s t r y , 10_,  211.  S c r a u p , H.W., G r e e n , M., and K u r l a n d , C.G., G e n e t i c s , 109, 193 (1970).  212.  L o d i s h , H.F.,  213.  Adams, J.M., J e p p e s e n , P.G.N., Sanger, F., and B., N a t u r e , 223, 1009 (1969).  214.  N i c h o l s , J.N.,  215.  L o d i s h , H.F., and R o b e r t s o n , H.D., C o l d S p r i n g Harbor Sym. Quant. B i o l . , 34, 655 (1969).  216.  S t e i t z , J.A., 824 (1970).  217.  D a r n e l l , J.E., B a c t e r i o l . Reviews,  218.  N i r e n b e r g , N.W., i n S.P. C o l o w i c k and N.O. K a p l a n ( E d i t o r s ) , Methods i n Enzymology, V o l . V I , Academic P r e s s , New Y o r k , 1963, p. 17.  219.  I w a s a k i , K., S a b o l . , S., Wahba, A . J . , and Ochoa, S., Biochem. B i o p h y s . , 125, 542 (1968).  220.  L u c a s - L e n a r d , J . , and Lipmann, F., P r o c . Nat. Acad. S c i . U.S.A., 5_5, 1562 (1966).  221.  D a v i s , B . J . , Ann. N.Y.  222.  R i c h a r d s , E.G., C o l l , J.A., Biochem., 12, 452 (1965).  223.  Peacock, A . C , (1967) .  and Dingman, C.W.,  224.  Peacock, A . C , (1968) .  and Dingman, C J . , B i o c h e m i s t r y , 1_,  Biochim.  and Hunt, J.A., N a t u r e , 221, 1118  and K u r l a n d , C.G.,  N a t u r e , 226, 705  N a t u r e , 225, 147  Dube, S.K.,  Mol.  (1969).  747  517 Gen.  (1970). Barrell,  (1970).  and R u d l a n d , P.S., 3_2, 262  Nature,  226,  (1968).  Acad. S c i . , 121, 404 and G r a t z e r , W.B.,  Arch.  (1964). Anal.  B i o c h e m i s t r y , 6_,  1818 668  158 225.  Dingman, C.W., (1968) .  and Peacock., A . C , B i o c h e m i s t r y , 1_, 659  226.  Cannon, M., K r u g , R., and G i l b e r t , W., J . M o l . 7, 360 (1963).  227.  M e s e l s o n , M., Nomura, M. , B r e n n e r , S. , D a v e r n , C , and S c h l e s s i n g e r , D. , J . M o l . B i o l . , 9_, 696 (1964) .  228.  G i l l a m , I . , M i l l w a r d , S., B l e w , D. von T i g e r s t r o m , M., Wimmer, E. , and T e n e r , G.M. , B i o c h e m i s t r y , 6_, 3043 (1967).  229.  L e i s , J.P., and K e l l e r , E.B., B i o c h e m i s t r y , 10, 889 (1971).  230.  H a l b r e i c h , A., and R a b i n o w i t z , M., P r o c . N a t . Acad. S c i . U.S.A. , 68,' 294 (1971) .  231.  D u n o f f , J.S., and M a i t r a , U., P r o c . N a t . Acad. S c i . U.S.A., £ 8 , 318 (1971).  232.  J o s t , J.P., and Bock, R.M., J . B i o l . Chem., 244, 5866 (1969) .  233.  Beaudet, A.L., and Caskey, C.T., P r o c . N a t . Acad. S c i . U.S.A. , 6_8, 619 (1971) .  234.  M i s k i n , R., Zamir, A., and E l s o n , D., J . M o l . B i o l . , 54, 355 (1970).  235.  S m i t h , A.E., and M a r c k e r ,  236.  Brown, J.C., and S m i t h , A.E., N a t u r e , 226, 610 (1970).  237.  Caskey, C.T., Beaudet, A.L., and N i r e n b e r g , M., J . M o l . B i o l . , 37, 99 (1968).  238.  G u p t a , N.K., J . B i o l . Chem., 243, 4959  239.  M a r s h a l l , R.E., Caskey, C T . , and N i r e n b e r g , M., S c i e n c e , 155, 820 (1967).  240.  S k o g e r s o n , L., and M o l d a v e , K., A r c h . Biochem. 125, 497 (1968).  241.  B l a c k , D.D., and G r i f f i n , A . C , Cancer Res., (1970).  242.  K l i n k , F., Kramer, G., Nour, A.M., and P e t e r s e n , K.G., Biochem. B i o p h y s . A c t a , 134, 360 (1967).  243.  R i c h t e r , D., and K l i n k , F., B i o c h e m i s t r y , 6, 3569  Biol.,  K.A., N a t u r e , 226, 607 (1970).  (1968).  Biophys.,  3j0, 1281  (1967).  159  244.  F u k a m i , H., a n d I m a h o r i , K., P r o c . N a t . A c a d . S c i . U.S.A., 68, 570 ( 1 9 7 1 ) .  245.  L e w i n , B., N a t u r e ,  246.  Galibert, M., B u l l .  247.  J o r d a n , B.R., F o r g e t , B.G., a n d M o n i e r , R., J . M o l . B i o l . , 55, 407 ( 1 9 7 1 ) .  248.  J o r d a n , R.R.,  249.  L e v i n , J . G . , a n d N i r e n b e r g , M., J . M o l . B i o l . , (1968).  250.  L e v i n , J.G., J . B i o l .  251.  H o a g l a n d , M.B., a n d C o o n l y , L . T . , P r o c . N a t . A c a d . S c i . U.S.A., 46, 1554 ( 1 9 6 0 ) .  252.  T a k a n a m i , M. , B i o c h i m . B i o p h y s . A c t a , 55_, 132  253.  Yu, C.T., a n d Z a m e c n i k , P . C , B i o c h i m . 45, 148 ( 1 9 6 0 ) .  254.  P r e i s s , J . , D i e c k m a n n , M., 236, 1748 ( 1 9 6 1 ) .  a n d B e r g , P., J . B i o l .  Chem.,  255.  N i r e n b e r g , M., a n d L e d e r , P., S c i e n c e , 145, 1399  (1964).  256.  H a r d e s t y , B., A r l i n g h a u s , R., S h a e f f e r , J . , a n d S c h w e e t , R. , C o l d S p r i n g H a r b o r Sym. Q u a n t . B i o l . , 2j}, 215 (1963).  257.  S t a n l e y , W.M., J r . , S a l a s , M., Wahba, A . J . , a n d O c h o a , S., P r o c . N a t . A c a d . S c i . U.S.A., 5 £ , 290 ( 1 9 6 6 ) .  258.  O f e n g a n d , J . , a n d H i n e s , C., J . B i o l . (1969) .  259.  S h i m i z u , N., H a y a s h i , H., a n d M u i r a , K., J . B i o c h e m . (Tokyo) , £ 7 , 373 (1970) .  260.  W a r n e r , J.R., a n d R i c h , A., P r o c . N a t . A c a d . S c i . U.S.A., 51, 1134 ( 1 9 6 4 ) .  261.  T a k a n a m i , M., B i o c h i m . B i o p h y s . A c t a , 6 1 , 432  262.  W e t t s t e i n , F.O., a n d N o l l , (1965).  263.  G i l b e r t , W.,  227, 1009  (1970).  F . , L a r s e n , C. J . , L e l o n g , J . C , a n d B o i r o n , S o c . Chim. B i o l . , £ 8 , 21 ( 1 9 6 6 ) .  J . Mol. B i o l . ,  5_5, 423  (1971).  Chem., 245, 3195  J . Mol. B i o l . ,  (1970).  (1962).  Biophys. Acta,  Chem., 244, 6241  H., J . M o l . B i o l . ,  6, 389  34, 467  (1963).  (1962). 11, 35  160 264.  E l s o n , D. , B i o c h i m . B i o p h y s . A c t a , 53_, 232  (1961).  265.  E l s o n , D., B i o c h i m . B i o p h y s . A c t a , 6 1 , 460  (1962).  266.  Oppenheim, J . , S c h e i n b u k s , J . , B i a v a , C., a n d M a r c u s , L . , B i o c h i m . B i o p h y s . A c t a , 161, 386 ( 1 9 6 8 ) .  267.  G o n z a l e z , N.S., G o l d e n b e r g , S.H., a n d A l g r a n a t i , I.D., B i o p h y s . A c t a , 166, 760 ( 1 9 6 8 ) .  268.  S c h e p s , R., Wax, R., a n d R e v e l , M., B i o c h i m . A c t a , 232, 140 ( 1 9 7 1 ) .  269.  I g a r a s h i , K., a n d K a j i , (1970).  270.  v o n D i g g e l e n , O.P., H e i n s i u s , H.L., K a l o u s e k , B o s c h , L . , J . M o l . B i o l . , 55, 277 ( 1 9 7 1 ) .  271.  S c h r e i e r , N.H., a n d N o l l ,  272.  G i l b e r t , W.,  273.  M o s t e l l e r , R.D., C u l p , W.J., a n d H a r d e s t y , B., J . B i o l . Chem., 243, 6343 ( 1 9 6 8 ) .  274.  Muench, K.H., a n d B e r g ,  275.  Muench, K.H., a n d S a f i l l e , (1968) .  276.  C u l p , W., M o r r i s e y , J . , a n d H a r d e s t y , B., B i o c h e m . B i o p h y s . R e s . Commun., 4 £ , 777 ( 1 9 7 0 ) .  277.  S m i t h , D.W.E., a n d McNamara, A . L . , S c i e n c e , 171, 577 (1971).  278.  B u r k a r d , G. , G u i l l e m a n t , P., a n d W e i l , J . H . , B i o c h i m . B i o p h y s . A c t a , 224, 184 ( 1 9 7 0 ) .  279.  Muench, K.H., a n d B e r g , P., i n G.L. C a n t o n i a n d D.R. Davies ( E d i t o r s ) , Procedures i n Nucleic A c i d Research, H a r p e r a n d Row, New Y o r k , 1966, p . 375.  280.  S h e a r n , A., a n d H o r o w i t z , N.H., B i o c h e m i s t r y , 8, 295 (1969) .  281.  S t a n l e y , W.H., a n d Wahba, A . J . , i n L . G r o s s m a n a n d K. Moldave ( E d i t o r s ) , Methods i n Enzymology, V o l . X I I , A c a d e m i c P r e s s , New Y o r k , 196 7, p . 524.  Biophys.  A., E u r . J . B i o c h e m . , 14, 41  H., N a t u r e ,  J . Mol. B i o l . ,  6, 389  F., and  229, 128  (1970).  (1963).  P., B i o c h e m i s t r y , 5_, 970  (1966).  P.A., B i o c h e m i s t r y , 1_, 2799  161  282.  H i l l , W.E., Anderegg, J.W., and yan Holde, K.E., J . Mol. Biol., 5 3 , 107 ( 1 9 7 0 ) .  283.  Bock, R.M., and C h e r a y i l , J.D., i n L. Grossman and K. Moldave (Editors), Methods i n Enzymology, V o l . XII, Academic Press, New York, 1 9 6 7 , p. 6 3 8 .  284.  Heppel, L.A., i b i d . , V o l . XII, 1 9 6 7 , p. 3 1 6 .  285.  K u r i k i , Y. , and K a j i , A., J . Mol. B i o l . ,  286.  Comb, D..G. , and Zehavi-Willner, T. , J . Mol. B i o l . , 2 3 , 441  2_5, 4 0 7  (1967).  (1967).  287.  Sarkar, N., and Comb, D.G., J . Mol. B i o l . ,  39_, 3 1  288.  Comb, D.G. , and Sarkar, N. , J . Mol. B i o l . ,  25_, 3 1 7  289.  Reynier, M., and Monier, R., B u l l . Soc. Chim. B i o l . , 5 0 , 1583  (1969). (1967).  (1968).  290.  Nirenberg, M., and Leder, P., Science,  291.  Bartz, J . , S o i l , D., Burrows, W.J., and Skoog, F., Proc. Nat. Acad. S c i . U.S.A., 6 7 , 1 4 4 8 ( 1 9 7 0 ) .  292.  Roy, K.L., Bloom, A., and S o i l , D., p r e p r i n t .  293.  Gillam, I.C., and Tener, G.M., i n L. Grossman and K. Moldave (Editors), Methods i n Enzymology, V o l . XX, Part C, Academic Press, New York, 1 9 7 1 , p. 5 5 .  294.  Stephenson, M.L., and Zamecnik, P.C., i n L. Grossman and K. Moldave (Editors), Methods i n Enzymology, V o l . XII, Part A, Academic Press, New York, 1 9 6 7 , p. 6 7 0 .  295.  Roy, K.L., and S o l i , D., Biochem. Biophys. Acta, 1 6 1 , 572  1 4 5 , 1399  (1964).  (1968).  296.  Jardetsky, O., and J u l i a n , G.R., Nature,  297.  S p i r i n , A.S., Kiselev, N.A., Shakulov, R.S., and Bogdanov, A.A., Biokhimiya, 28, 7 6 5 ( 1 9 6 3 ) .  298.  Elson, D., i n D.B. Roodyn (Editor), Enzyme Cytology, Academic Press, New York, 1 9 6 7 , p. 4 0 7 .  299.  Szer, W. , Biochem. Biophys. Res. Commun.,  300.  Egami, F., and Nakamura, K., i n A. K l e i n z e l l e r , G.F. Springer and H.G. Wittmann (Editors), Molecular Biology, Vol. 6 , Academic Press, New York, 1 9 6 9 .  2 0 1 , 397  35_ 6 5 3  (1964).  (1969).  162 301.  Neu, H . C , and H e p p e l , L.A. , J . B i o l . Chem., 239, 3893 (1964).  302.  D e l i h a s , N. , Biochem. B i o p h y s . Res. Commun., 3_9, 905 (1970) .  303.  Ehresmann, C., and E b e l , J.P., E u r . J . Biochem., 1 3 , 577 (1970).  304.  G u p t a , S.L., Chen, J . , S c h a e f e r , L., L e n g y e l , P., and Weissman, S.M., Biochem. B i o p h y s . Res. Commun., 39, 883 (1970).  305.  K u e c h l e r , E., and R i c h , A., N a t u r e , 225, 920 (1970).  306.  K u r l a n d , C.G., J . M o l . B i o l . , 18, 90 (1966).  307.  C r i c k , F.H.C, J . M o l . B i o l . , 19, 548 (1966).  308.  D i x o n , G.H., p e r s o n a l  309.  G i l m o u r , S., and D i x o n , G.H., p e r s o n a l  communication. communication.  163 APPENDIX 50 y l o f ^ C - P h e  (42 yg and 0.1 mCi/ml) was d i l u t e d w i t h  10 0 y l d i s t i l l e d w a t e r . each a s s a y .  5 y l o f t h i s m i x t u r e was used f o r  Two c o n t r o l s were used t h r o u g h o u t .  One con-  t r o l c o n t a i n e d a l l t h e assay components e x c e p t p o l y U w h i l e t h e o t h e r c o n t r o l c o n t a i n e d a l l t h e assay components e x c e p t WRib.  The l e v e l o f r a d i o a c t i v i t y o b s e r v e d i n b o t h  was t h e s a m e — a p p r o x i m a t e l y  200 cpm.  a c t i v i t y s e r v e d as t h e background  controls  This l e v e l of radio-  and was s e t a t one.  50 y l o f '*C-amino a c i d m i x t u r e (1 mCi/ml) was d i l u t e d w i t h 1  10 0 y l d i s t i l l e d w a t e r . used f o r each a s s a y . 52-53 was added. tRNA.  5 y l o f t h i s d i l u t e d m i x t u r e was  Enzyme p r e p a r e d as d e s c r i b e d on pages  A l l tubes c o n t a i n e d t h e same amount o f  Two c o n t r o l s were used t h r o u g h o u t .  One c o n t r o l con-  t a i n e d a l l t h e assay components e x c e p t tRNA (tRNA c o n t r o l ) w h i l e t h e o t h e r c o n t r o l c o n t a i n e d a l l t h e assay components e x c e p t enzyme. background  S i n c e t h e tRNA c o n t r o l gave t h e h i g h e s t  l e v e l o f r a d i o a c t i v i t y , t h i s background was  sub-  t r a c t e d from each o f t h e sample t u b e s . The assay system c o n t a i n e d a l l t h e components p r e v i o u s l y d e s c r i b e d on page 51 i n M a t e r i a l s and Methods. tubes c o n t a i n e d t h e same amount o f tRNA. d e s c r i b e d on pages 52-53 was used 1  A l l the  Enzyme p r e p a r e d as  : 5 y l o f the s p e c i f i c  ''C-amino a c i d was added t o t h e p a r t i c u l a r t u b e .  The  c o n c e n t r a t i o n and s p e c i f i c a c t i v i t y o f t h e "'C-amino a c i d s 1  164 used a r e g i v e n below:  Ala Arg Asn Asp Gly Glu His He Leu Lys Met Phe Pro Ser Thr Trp Tyr Val  pg/ml  mCi/ml  76.0 37.0 282.0 86 .0 56 .0 71.0 1.2 55.0 52.0 30 .0 1100 .0 42.0 31.0 84.0 37.0 900 .0 48.0 63.0  0 .10 0 .05 0 .10 0 .10 0 .10 0 .10 0.05 0.10 0 .10 0 .05 0 .10 0 .10 0 .05 0 .10 0.05 0 .10 0 .10 0 .10  Two c o n t r o l s were u s e d f o r each s p e c i f i c amino a c i d t e s t e d . One c o n t r o l c o n t a i n e d a l l t h e assay  components e x c e p t  tRNA  (tRNA c o n t r o l ) w h i l e t h e o t h e r c o n t r o l c o n t a i n e d a l l t h e assay  components e x c e p t enzyme.  S i n c e t h e tRNA c o n t r o l  gave t h e h i g h e s t b a c k g r o u n d l e v e l o f r a d i o a c t i v i t y ,  this  b a c k g r o u n d was s u b t r a c t e d from t h e sample tube c o n t a i n i n g tRNA.  The e x p e r i m e n t a l c o n d i t i o n s a r e s i m i l a r t o t h o s e  used by K e l l e r  (229) and Muench and B e r g  (279).  

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