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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., University of Manitoba, 1964 M.Sc, University of Manitoba, 1967 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY In The Department of Biochemistry We accept t h i s thesis as conforming to the required standard The University of B r i t i s h Columbia Ju 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 requirements f o r an advanced degree at the U n i v e r s i t y of B r i t i s h Co lumbia , I agree that the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r re ference and study . I f u r t h e r agree t h a t permiss ion fo r e x t e n s i v e copying o f t h i s t h e s i s f o r s c h o l a r l y purposes may be granted by the Head of my Department or by h i s r e p r e s e n t a t i v e s . I t i s understood that copying o r p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l ga in s h a l l not be a l lowed wi thout my w r i t t e n p e r m i s s i o n . \ 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 i ABSTRACT Ribosomes were readi l y prepared by various procedures from c o l i B c e l l s grown to the mid-log or late log phase. The a b i l i t y of these ribosomes to support protein synthesis varied with the method of preparation. The low molecular weight RNA (LMWRNA) bound to these ribosomes was studied and i t , too, vari e d with the type of preparation. A f t e r these i n i t i a l s tudies, the procedure which e n t a i l e d seven extractions with 0.5-1.0 M NH^Cl i n the presence of varying levels of magnesium concentration was used i n a l l further experiments for the preparation of ribosomes (WRib). Attempts were made to remove a l l the tRNA bound to WRib. Treatment with H I O i , resulted i n a complete disruption of ribosomal structure and was abandoned. Puromycin (PM) tr e a t -ment f a i l e d to remove a l l the bound tRNA but resulted i n a more active preparation. Incubation of these PM-treated WRib with 0.1 mM Mg + + not only removed a l l the bound tRNA but also resulted i n i n a c t i v e preparations. Treatment of the WRib with 0.1 mM Mg + + alone gave i d e n t i c a l r e s u l t s . Ribosomal subunits were found to be devoid of bound tRNA. Attempts to subst i t u t e these subunits i n a protein-synthesizing system i n place of whole ribosomes (WRib) f a i l e d . The LMWRNA bound to WRib was fractionated by a number of techniques - Sephadex G-100 chromatography, DEAE-Sephadex A-50, and preparative gel electrophoresis. The o p t i c a l density patterns of the fractionations showed only small d i f f e r e n c e s , however, acrylamide gel electrophoresis studies i i of the peak fr a c t i o n s indicated that chromatography on Sepha-dex G-100 was the method of choice f o r the separation of LMW-RNA. The tRNA bound to WRib was f u l l y characterized. c o l i WRib have bound tRNA which has acceptor a c t i v i t y f o r a l l the amino acids tested. The amount of charging varied from one tRNA species to another - those for tryptophan and methionine were bound i n the l a r g e s t amounts. The s i g n i f i c a n c e of these r e s u l t s cannot, as yet, be explained. The t o t a l amount of tRNA bound to WRib amounted to approximately 1.3-1.7% of the t o t a l ribosomal RNA (rRNA). S i m i l a r l y the amount of 5S RNA bound represented 1.5-2.8% of the t o t a l rRNA. These percent-ages are equivalent to 0.9-1.3 molecules of tRNA bound per molecule of 5S RNA. Another species of rRNA, 4.5S RNA was found bound to the WRib but i n small amounts. Most of the 5S RNA preparations contained t h i s RNA. The tRNA bound to WRib, prepared from a pyrimidine-requiring mutant (ATCC13135) grown i n the presence of 3H-u r a c i l , was exchanged with unlabeled tRNA. After exchange 5.7 molecules of tRNA were bound per molecule of 5S RNA and v i r t u a l l y a l l of the labeled tRNA had been removed. These exchanged WRib were no longer active i n a protein-synthesizing system. The tRNA from these WRib was fractionated on a BD-c e l l u l o s e column. The r a d i o a c t i v i t y was evenly spread throughout the s a l t gradient but a peak was i s o l a t e d i n the ethanol gradient. The r a d i o a c t i v i t y i n t h i s peak region may be accounted for by the presence of 4.5S RNA. The r e s u l t s I l l suggest that a l l tRNAs are bound to 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 reported i n the l i t e r a t u r e of the non-existence of a s p e c i f i c chain-termin-ating tRNA. i v ACKNOWLEDGEMENTS I wish to thank my research supervisor Dr. Gordon M. Tener for enthusiastic support, valuable suggestions and discussions during the course of this work. My associations with the members of this laboratory have been most helpful and I wish to acknowledge the debt I owe to them. V TABLE OF CONTENTS PAGE Abstract i Acknowledgements i v Table of Contents v L i s t of Tables and Charts v i i i L i s t of Figures i x Abbreviations x i i L i s t of Buffers x i i i Dedication x i v Introduction 1 The Ribosome 1 Ribosomal RNA 3 Heterogeneity of Ribosomal RNA 7 Ribosomal Proteins 8 Stoichiometry of Ribosomal Proteins 9 RNA-Protein Interaction and the Internal 12 Organization of the Ribosome Binding of tRNA to Ribosomes 14 (a) Non-specific Binding of Free tRNA 14 (b) Non-enzymatic S p e c i f i c Binding of 17 Aminoacyl-tRNA (c) Enzyme-Specific Binding of Aminoacyl- 22 tRNA (d) Summary 22 The Ribosome and Protein Synthesis 24 Release Factors and the Mechanism of 28 Termination Ribosomal Structure and Function 33 v i PAGE Outline of the Problem 43 Thesis Proposal 44 Materials and Methods 45 Chemicals 45 Preparation of E^ c o l i B 45 Preparation of E^ c o l i B Mutant 12632 46 Preparation of E^ c o l i B. Ribosomes 48 Assay f o r Ribosomal A c t i v i t y and Amino 50 Acid Incorporation Assay f o r Amino Acid Acceptor A c t i v i t y 51 Preparation of E^ c o l i Aminoacyl-tRNA 52 Synthetases (a) Treatment DEAE-Cellulose Column 52 (b) Treatment on Sephadex G-25 Column 53 Preparation of Benzoylated DEAE-Cellulose 54 Treatment of Ribosomes with Puromycin 55 Treatment of Ribosomes with Periodate 56 A n a l y t i c a l Polyacrylamide Gel E l e c t r o -phoresis 56 Preparative Polyacrylamide Gel E l e c t r o - 60 phoresis Preparation of 30S and 50S Subunits from 62 Ribosomes Preparation of CsCl Gradient 63 I s o l a t i o n of T o t a l RNA from Ribosomes 63 Preparation of High and Low Molecular 64 Weight RNA from Ribosomes Results 68 v i i PAGE The A c t i v i t y o f Ribosome P r e p a r a t i o n s 6 8 Attempts t o Remove Bound tRNA from 70 Ribosome (a) Puromycin 70 (b) P e r i o d a t e 72 (c) D i a l y s i s a g a i n s t 0.1 mM Magnesium 75 S t u d i e s on the 30S and 50S Ribosomal 75 Subunits C h a r a c t e r i z a t i o n of the Ribosomes 79 Other Techniques Used t o F r a c t i o n a t e 91 Low M o l e c u l a r Weight 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 91 (b) DEAE-Sephadex Chromatography 96 F r a c t i o n a t i o n of Ribosomal RNA by 100 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 Exchange Experiments 104 D i s c u s s i o n 119 B i b l i o g r a p h y 146 Appendix 16 3 v i i i LIST OF TABLES AND CHARTS TABLE PAGE I Amino acid acceptor a c t i v i t y of ribosomes 67 prepared by d i f f e r e n t methods II D i s t r i b u t i o n of RNA i n E ^ c o l i ribosomes 82 III S p e c i f i c amino acid acceptor a c t i v i t y of 85 tRNA bound to E^ c o l i ribosomes IV D i s t r i b u t i o n of low molecular weight RNA i n 88 E. c o l i ribosomes i s o l a t e d by Sephadex G-100 chromatogr aphy V Exchange of l a b e l l e d tRNA bound to c o l i 107 ribosomes with unlabelled tRNA VI D i s t r i b u t i o n of l a b e l l e d 3H-RNA i n E^ c o l i 112 ribosomes a f t e r exchange with unlabelled tRNA CHART I Preparation of E^ c o l i ribosomes 47 II Procedures for preparing c o l i ribosomes 66 ix LIST OF FIGURES FIGURE PAGE 1 Photograph of polyacrylamide gel e l e c t r o - 69 phoresis pattern of low molecular weight RNA obtained from d i f f e r e n t preparations of E^ _ c o l i ribosomes 2 Photograph of polyacrylamide gel e l e c t r o - 71 phoresis patterns of low molecular weight RNA obtained from puromycin-treated E^ _ c o l i ribosomes 3 Photograph of polyacrylamide gel e l e c t r o - 73 phoresis patterns of low molecular weight RNA obtained from magnesium-treated E.  c o l i ribosomes 4 Sedimentation pattern of E^ c o l i ribosomes 74 on a 10-30% discontinuous sucrose gradient 5 Photograph of polyacrylamide gel e l e c t r o - 76 phoresis patterns of low molecular weight RNA obtained from E^ c o l i ribosomal subunits 6 Sedimentation pattern of E^ c o l i ribosomal 77 subunits i n a CsCl s o l u t i o n 7 E l u t i o n pattern of commercial E^ c o l i tRNA 78 from Sephadex G-100 8 Photograph of polyacrylamide gel e l e c t r o - 80 phoresis patterns 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 previously on a Sephadex G-100 column 9 E l u t i o n pattern of t o t a l ribosomal RNA from 81 E. c o l i on Sephadex G-100 10 Photograph of polyacrylamide gel e l e c t r o - 84 phoresis patterns of peaks II and III as designated i n Table II 11 E l u t i o n pattern of the low molecular weight 86 RNA bound to E^ c o l i ribosomes from Sephadex G-100 12 Photograph of polyacrylamide gel e l e c t r o - 89 phoresis patterns of the peak f r a c t i o n of each peak region of Figure 11 X FIGURE PAGE 12a Chromoscan tra c i n g of a mixture of f r a c t i o n s 90 116 and 132 (peaks II and III) from Figure 11 13 Electrophoresis pattern on a 10% preparative 92 polyacrylamide gel of 5 mg of commercial E. c o l i tRNA 14 Electrophoresis pattern of 100 mg of commer- 93 c i a l E^ c o l i tRNA on a 10% preparative polyacrylamide gel 15 Chromatography of commercial c o l i tRNA 94 on DEAE-Sephadex A-50 16 Photograph of polyacrylamide gel e l e c t r o - 95 phoresis patterns of the peak f r a c t i o n of each peak region of Figure 15 17 Electrophoretic pattern of the low molecular 97 weight RNA from E^ c o l i ribosomes i n a 10% preparative polyacrylamide gel 18 Photograph of polyacrylamide g e l e l e c t r o - 98 phoretic patterns of the peak f r a c t i o n of each peak region of Figure 17 19 Spectrum of f r a c t i o n 26 (peak I) of Figure 17 99 20 The e f f e c t of pH on the spectrum of f r a c t i o n 101 26 (peak I) of Figure 17 21 Growth and pH curves of an E^ c o l i pyrimi- 103 dine r e q u i r i n g mutant (ATCC 13135) 22 Displacement of l a b e l l e d tRNA bound to E^ 105 c o l i ribosomes 23 Exchange of l a b e l l e d tRNA bound to E^ c o l i 10 8 ribosomes with unlabelled tRNA 24 E l u t i o n pattern from Sephadex G-100 of the 110 low molecular weight RNA bound to l a b e l l e d E. c o l i ribosomes a f t e r exchange with unlabelled tRNA 25 Photograph of polyacrylamide gel e l e c t r o p h o r e s i s l l l patterns of various f r a c t i o n s from Figure 24 x i FIGURE PAGE 26 Chromatography on BD-cellulose of 4S RNA 114 bound to E ^ c o l i ribosomes a f t e r exchange with unlabelled tRNA 27 Chromatography on BD-cellulose of 4S RNA 115 normally bound to E_^ c o l i ribosomes 28 Chromatography on BD-cellulose of commercial 117 E. c o l i tRNA x i i ABBREVIATIONS A l l abbreviations and terminology used i n t h i s thesis are i n accordance with those normally accepted by the J. B i o l . Chem., 246, 4 (1971) and only those abbreviations not l i s t e d i n t h i s journal are shown below. fMet aa-tRNA tRNA Met m or f DF R factor TEMED WRib PM ATA PPO dimethyl-POPOP DMAPN TCA A2 6 0 A26O unit Bis Mg(OAc) 2BD-cellulose formylmethionine Aminoacyl-tRNA A s p e c i f i c member of the group of tRNAs which accept methionine. D i s s o c i a t i o n Factor Release Factor N,N,N',N'-Tetramethylethylenediamine Washed Ribosomes Puromycin A u r i n t r i c a r b o x y l i c Acid 2,5-diphenyloxazole 1,4-bis-2-(4-methyl-5-phenyloxazolyl)-benzene 3 1-dimethylaminopropionitrile T r i c h l o r o a c e t i c Acid Absorbance at 260 nm One A2 6 0 unit i s that amount of material which when dissolved i n one ml of solvent w i l l give an absorbance of one i n a c e l l with a l i g h t path of 1 cm. N,N'-Methylenebisacrylamide magnesium acetate Benzoylated DEAE-cellulose x i i i LIST OF BUFFERS Buffer Components A lOmM T r i s , ethanol, lOmM Mg(OAc) 2 pH 7.8 , lOmM NHifCl, lOmM mercapto-B lOmM T r i s , O.lmM Mg(OAc) 2 , 0.5-1.0M NHijCl, pH 7.4 C lOmM T r i s , ImM Mg(OAc) 2, 0.5-1.0M NHi»Cl, pH 7.4 D lOmM T r i s , 5mM Mg(OAc) 2, 0.5-1.0M NH^Cl, pH 7.4 E 10 mM T r i s , lOmM Mg(OAc) 2 , 0.5-1.0M NHi»Cl, pH 7.4 F lOmM T r i s , lOmM Mg(OAc) 2 , 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 cysteine, pH 7.4 I lOmM T r i s , O.lmM Mg(OAc) 2 , pH 7.6 K 5mM T r i s , O.lmM Mg(OAc) 2 l pH 7.3 L lOmM PH T r i s , 7.6 lOmM Mg(OAc) 2 , 0.5% sodium dodecyl s u l f a t e , M lOmM T r i s , 5mM Mg(OAc) 2, 250mM NH4CI, pH 7.8 N lOmM T r i s , lOmM Mg(OAc) 2 , 1.0M NH^Cl, pH 7.8 x i v DEDICATION To my parents who i n s t i l l e d me with the drive and incentive, to my dearest Bev who gave me encouragement, confidence and love and to Stephanie for p r i o r i t y lessons 1 INTRODUCTION The amino acid sequence of a particular protein i s specified by the sequence of nucleotides in a particular seg-ment of the deoxyribonucleic acid (DNA). In the transcription step the DNA i s transcribed into a ribonucleic acid (RNA) intermediate called messenger RNA (mRNA), which has a ribo-nucleotide sequence complementary to that of the deoxyribonu-cleotide sequence of one of the strands of the DNA serving as template (64). The translation steps follows. The mRNA becomes attached to cytoplasmic ribonucleoprotein particles (ribosomes) which are the sites of protein synthesis, and there i t determines the order of linkage of amino acids into a specific protein (65-67) . During translation a group of three adjacent nucleotides in the mRNA (codon) specifies which amino acid i s to be linked to the growing peptide chain. There are 64 possible codon triplets and 61 of these specify a particular amino acid (68). The other three codons, UAA, UAG and UGA are called nonsense codons since they act as chain terminators which signal the end of a genetic message. Each amino acid i s joined specifically to one of the codon-recognizing molecules known as transfer RNA (tRNA). Each tRNA molecule has i t s own t r i p l e t of bases, called an anti-codon, that recognizes the relevant codon on the mRNA by pairing bases with i t . The Ribosome Zamecnik and his co-workers established the central role 2 of ribosomes i n protein synthesis (although earlier cytologi-cal studies had shown ribosomes to be the site of protein synthesis), and in addition, discovered most of the components involved in in vitro 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 for the proteins synthesized on the ribosomes. 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 isolation and characterization of ribosomes were initiated (2-5) . These studies were done on ribosomes from c o l i and established the following basic information : (a) Ribosomes isolated and purified in the presence of 10 mM Mg + + have a sedimentation coefficient of 70S. After treatment with puromycin which removes nascent peptides, these ribosomes dissociate into 3OS and 50S subunits upon lowering Mg + + concentration (1 mM or less) (2,6); however, i f nascent peptide is present, complete removal of Mg + + with EDTA is required (69). (b) The 50S and 30S ribosomal subunits have a particle weight of 1.8 x 106 and 0.85 x 10 6 daltons respectively (6). (c) Both 50S and 30S subunits contain about 2/3 RNA and 1/3 protein. (d) The 50S subunit contains one molecule of RNA, 23S RNA (The presence of one molecule of 5S RNA was discovered later (55)) , and the 30S subunit contains one molecule of RNA, 16S RNA (7). 3 Subsequent progress in the study of ribosomes, however, lagged far behind progress in other areas of molecular biology. This was partly due to the discovery of mRNA which diverted attention from ribosomes, and partly due to the d i f f i c u l t i e s caused by the structural complexity of ribosomes. For example, 30S subunits contain about 20 different proteins and 50S subunits contain about 30 to 35 different proteins. 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. Moreover, an active role for ribosomes in the codon-anticodon recognition process was suggested (8). Thus serious interest in the ribosome was revived and the l a s t several years have witnessed rapid progress in 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 106 and one molecule of 5S RNA with a molecular weight of 4 x 10" (7, 42, 44). Since the molecular weight of 23S RNA i s about twice that of the 16S RNA, there have been frequent claims that the 23S RNA i s a dimer of a "16S" RNA molecule which i s identical or very similar to the 16S RNA molecule found in 30S ribosomal subunits. Several observations originally supported this idea (7, 45-47). 4 One can now exclude this claim. The 23S and 16S rRNAs have been shown to be different in base composition (44), base sequence as judged by DNA-RNA hybridization (48) , oligo-nucleotide patterns obtained after enzymatic digestion (49), methylated oligonucleotides obtained after Ti RNase digestion (47) and in their 5'-terminal sequences (50). It was also found that the "16Sn RNA prepared from 23S RNA according to the method of Midgley (45) could not replace 16S rRNA in the reconstitution of 30S ribosomal subunits. Thus, i t was unlikely 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 is 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, careful studies done by Stanley and Bock (127) revealed no non-covalent bond in the 23S rRNA molecule. Finally Leppla (52) measured the number of chain terminal bases in the 23S rRNA molecule and obtained results which were consistent with one chain terminus per 23S rRNA. I t therefore appears that the 23S rRNA is a single polynucleotide chain with a molecular weight of 1.1 x 10 6. Although the 23S rRNA i s a single polynucleotide chain, the work of Fellner and Sanger (47) strongly indicated that the molecule was made up of two sections which may be identi-cal or very similar in their base sequence. One of their 5 suggestions was that the 23S rRNA cistron had arisen by a "gene duplication" mechanism during evolution. Whether the possible existence of two identical or similar parts i s related to the functions of 23S rRNA (53) i s not clear. A related subject i s the problem of sequence homology between 16S and 23S rRNAs. Despite clear-cut evidence for a sequence d i f f e r -ence between E_^_ c o l i 16S and 23S rRNAs, DNA-RNA hybridization experiments have shown that 16S and 23S rRNAs compete for the same DNA sites to a great extent (54) . This suggests that DNA cistrons for both 16S and 23S RNA have evolved by gene duplication starting from a common gene. Alternatively, par t i a l sequence homology may reflect a common (unknown) function performed by parts of both 16S and 23S RNAs. On the other hand, DNA-RNA hybridization experiments done with Bacillus megaterium and Bacillus s u b t i l i s showed a complete lack of sequence homology (48) . I t is not clear i f the observed discrepancy was due to a difference i n techniques used or to the difference in bacterial species. Maturation of rRNAs in 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 spl i t t i n g a single large precursor molecule whereas in bacteria the two rRNAs are derived from two discrete precursors each only slightly larger than the mature species (206) . 6 The 50S ribosomal subunit contains one molecule of 5S RNA in addition to 23S rRNA (55). The 5S RNA does not accept amino acids and i s thus different from tRNA. I t i s not a random breakdown product of 16S or 23S rRNA but appears to be a genuine ribosomal component present in a l l 50S subunits of various origins. To date l i t t l e , i f any i s known of the functional role of 5S RNA although Kirtikar and Kaji (128) have shown that the addition of 5S RNA stimulated incorpora-tion of amino acids into protein directed by RNA from phage MS-2. Siddiqui and Hosokawa (131) have suggested that 5S rRNA may have a role in the specific binding of tRNA to ribo-somes. Raacke (132) has proposed a cloverleaf conformation for 5S RNAs. The model proposes three functions for 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 in a species-specific manner, (3) the binding of tRNA through specific base pairing and Mg bridges. Recently Jordan (248) has found that the most exposed part of the c o l i 5S RNA (as determined by Ti RNase cleavage) has a sequence complementary to the -GTUCG sequences found in a l l tRNAs thus far sequenced. Although this would appear to strengthen the case for a function of 5S RNA involving inter-action with tRNA in the ribosome, this sequence in tRNA is in the most protected loop. Therefore in order for interaction to occur the tRNA would have to undergo a conformatioal change which would involve ring opening. Other models have also been proposed (42, 133-135) . The 5S RNA from E_j_ c o l i 7 consists of 120 nucleotides and lacks methylated or unusual bases in contrast to other rRNAs or tRNAs (42) . The nucleo-tide sequence of 5S RNA from c o l i (42) and from mammalian KB cel l s (129) has been established, and i t is interesting to note that a part of the nucleotide sequence is duplicated in each of the two 5S RNA species. Preliminary studies also indicate that the sequence of 5S rRNA from two mouse c e l l lines i s the same as that found in human KB cells (136) . Recently the nucleotide sequence of Pseudomonas fluorescens 5S RNA has been elucidated and found to contain many similar-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 hybridization (56) . Recently Monier's group (130) demonstrated the presence in exponentially growing E. c o l i c e l l s of a precursor of 5S RNA. The precursor was longer by 1-3 nucleotides. A similar precursor was synthe-sized i n the presence of chloramphenicol (247). Heterogeneity of Ribosomal RNA DNA-RNA hybridization experiments have clearly shown that genes for 16S and 23S rRNA are present in multiple copies (137-140). For instance, Spadari and Ritossa (137) confirmed the existence of 6 genes for 23S rRNA and 6 for 16S rRNA for each Ej_ c o l i chromosome. Because of this redun-dancy, i t i s quite possible that the genes for 16S rRNA or those for 23S rRNA are not homogeneous and that there are several chemically different species of 16S rRNA and 23S rRNA 8 (46, 50, 57). At least three different l o c i for 5S RNA cistrons have been localized in E_j_ c o l i (141) . The question of the possible heterogeneity of rRNAs is important, since i t implies the heterogeneity of each of the ribosomal subunits and i s possibly related to some functional differentiation among ribosomes. On the other hand, the sequence analysis of methylated oligonucleotides of rRNA done by Fellner and Sanger (47) showed that many long oligonucleotides with unique base sequences and chain lengths up to eleven occurred in one, two or four moles per mole of RNA, and never in an amount less than one mole. Thus, both 16S and 23S rRNA from c o l i are largely homogenous, at least with respect to the base sequences around methylated nucleotides. Further extensive analysis of fragments of 16S rRNA performed by Fellner 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. I t should be noted that the base sequence of 5S RNA from c o l i has proved the homogeneity of this RNA species (42) , despite the multiplicity of cistrons for this RNA in the bacterial genome (48) . Ribosomal Proteins I t appears that the 30S ribosomal subunit of c o l i contains about 21 proteins. This has been confirmed by both Kurland's (33, 210) and Nomura's groups (38). The average 9 molecular weight i s 12,400 per protein. Traut and his co-workers (34) isolated 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 his co-workers concluded that the number of 50S proteins is 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). It is significant to note that there i s no protein common to both 50S and 30S particles. This conclusion was obtained by both chemical and immunologi-cal studies (33-36, 121-122, 124-126). The results favour the conclusion that there i s no extensive structural homology among ribosomal proteins (rProteins). Stoichiometry of Ribosomal Proteins I t has usually been assumed that the ribosome has a defined structure and that the ribosome population i s homo-geneous. The heterogeneity of rRNA and hence of ribosome populations was suggested by several investigators, but i t i s only now that Kurland's group has performed careful 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 this problem by Moore's group (35) showed that most (thirteen) of the ribosomal proteins existed in amounts corresponding to one copy per 30S particle, the conclusion was readily accepted that the ribosomes were homogeneous with respect to their 10 protein composition. However, l a t e r work by Kurland et a l . (36) did not agree with t h i s conclusion. They examined 21 ribosomal proteins and found that 12 of them were present i n amounts corresponding to about one copy per 30S p a r t i c l e , but that 8 other proteins were present i n amounts less than 0.7 copies per ribosome. There was no evidence which suggested that any 30S protein was present i n amounts corresponding to more than 1 copy per ribosome. They concluded that there are two kinds of proteins; one which they c a l l e d "unit protein" i s present i n a l l the i s o l a t e d 30S ribosomal p a r t i c l e s and the other which they c a l l e d " f r a c t i o n a l protein" i s present 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 . Discrepancies between Kurland's and Moore's laboratories were mostly i n the molecular weight values assigned to some of the proteins. Although work done by another group also favored the conclu-sion that a l l the ribosomal proteins existed i n stoichiometric amounts (37) , i t now appears that the conclusion obtained by Kurland's group i s correct, at l e a s t with respect to ribosomes obtained as i n v i t r o preparations. Recent investigations of molecular weights of 30S ribosomal proteins by Moore's group has now yielded data consistent with that obtained by Kurland and his collaborators (34). There are several other facts supporting the conclusion of a heterogeneous population of ribosomes. F i r s t , there i s a s t r i k i n g c o r r e l a t i o n between proteins c l a s s i f i e d as unit proteins by Kurland et al_. and proteins found by Nomura's group to be required for the 11 "physical assembly" of ribosomes. Such correlation 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 activity of 30S particles by incubating them with externally added 30S ribosomal proteins under the conditions optimum for reconsti-tution. Concomitant with this stimulation, they observed that some externally added proteins were incorporated into the particles and some proteins i n i t i a l l y present in the 30S particles were released into the medium (36). Although interpretation of the observed protein exchanges must await exact identification of these exchanged proteins, the observed facts are consistent with the conclusion that the isolated 30S particles are not f u l l y active and that part of the reason for the inactivity i s a deficiency in some ribosomal proteins in some of the 30S particles. An alternative explanation i s the steady-state hypothesis of Kurland (210) who postulates a functional cycle in which the complement of fractional proteins on a ribosome changes as a given ribosome proceeds through the different operational modes. One example of such a cycle would entail different sets of fractional proteins associated with the ribosomes during chain i n i t i a t i o n , propagation, termination and a rest mode. Some of the proteins which are involved in 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 in amounts corresponding to one copy per 50S particle. Traut et al^. (34) found that 31 of the 50S ribosomal proteins existed in stoichiometric amounts and only 2, or possibly 4, of the 50S ribosomal proteins existed in amounts that were much less than one copy per particle. Kurland's group has also failed to detect any significant heterogeneity of 50S ribosomal subunit populations so far. A major conclusion which has emerged from studies on ribosomal proteins i s that, because none of the proteins exists as more than one copy per particle, ribosomal subunits have no symmetry. This means that any model of ribosome function involving structural symmetry, for example, the presence of two or more identical sites 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 different ribosomal protein molecules (38,60). I t was found that the assembly reaction required the presence of a specific RNA. In the absence of rRNA, no particles resembling 30S ribosomal subunits were formed. Furthermore, neither 17S cytoplasmic rRNA from yeast nor "16S" RNA prepared from c o l i 23S rRNA could replace the 16S c o l i rRNA in the reconstitution. With these two RNAs no particle sedimenting at 30S was formed. Rat l i v e r 18S rRNA also could not replace 16S E. c o l i rRNA in the 13 reconstitution. These experiments clearly show that the rRNA-ribosomal protein interaction is specific and is important for the overall organization of ribosomal particles. Recently Nomura's group has been able to reconstitute 50S ribosomal subunits of Bacillus stearothermophilus (144) . They were able to show a partial requirement for 5S RNA in the reconstitution of functional 50S particles. Their results also indicated specificity of binding between the 23S rRNA and ribosomal proteins. Nomura's group has also shown that a l l mutational altera-tions discovered so far which inhibit 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 affect 30S assembly (145-146). They proposed that assembly of 50S ribosomal subunits somehow depended on simultaneous assembly of 30S ribosomal subunits in vivo, while the assembly of 30S particles was independent of 50S assembly. Experiments to date strongly suggest that single-stranded regions are important in the RNA-protein interaction, but do not exclude the possible additional involvement of helical regions in the interaction (234). The chemical basis of the specificity demonstrated in the RNA-protein interaction 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 detailed model of ribosome structure. The topological relationship of the many different molecular components is 14 unknown. Even the very elementary questions as to which pro-teins are on the surface and which proteins are buried inside, or which part of the rRNA molecule i s exposed on the surface of the ribosome, are not answered. However, i t is believed that at least a part of the rRNA i s exposed and thus the ribosomal strucutre i s drastically different from the common spherical virus structure in which the RNA is completely protected by an outer protein s h e l l . Miskin et a l . (234) suggest that a conformational change in a protein, a local rearrangement of a helic a l portion of RNA, or an alteration i n some protein-RNA association are some of the po s s i b i l i t i e s that can account for the transition between the active and inactive states of the ribosome (62, 63). A l l detailed structural models must be considered highly speculative at this time. Binding of tRNA to Ribosomes (a) Non-Specific Binding of Free tRNA Both tRNA and aa-tRNA were shown in early studies to attach to ribosomes (148, 251-252). The tRNA-ribosome complex is stable only in solutions of high Mg + + ion concentration. I t i s s t i l l unknown what forces are involved in holding the two components together in a stable complex. Presumably, magnesium bridges are formed not only between subunits but also between the tRNA-ribosome complex. The Mg + + ions tend to overcome the mutual repulsion of the phosphate groups of RNA to allow binding. Bound tRNA w i l l not wash off in high 15 Mg + + ion concentration but can e a s i l y be displaced by free tRNA from the surrounding medium (148) . This exchange i s not affected by charging tRNA with amino acids (249-250) , by temperature, or by puromycin or chloromphenicol (148) . The nonspecific a ssociation of tRNA and ribosomes takes place i n the cold and does not require the supernatant enzymes, GTP, ATP or an energy source other than the thermal energy of the reacting components. I f the terminal pCpCpA sequence of the tRNA i s damaged t h i s binding does not occur. I f the terminal adenosine i s removed by o x i d i z i n g deacylated tRNA with p e r i o -date followed by treatment with eyelohexylamine (253) , the a b i l i t y of the tRNA to bind to the ribosomes i s destroyed (148) . I f the terminal sequence i s reformed (254) by incubating the tRNA with the supernatant f r a c t i o n and ATP, or ATP and CTP, the a b i l i t y to bind i s p a r t i a l l y re-established (252) . The requirement f o r the i n t a c t pCpCpA terminus common to a l l tRNA molecules, indicates that the terminal sequence plays a decisive r o l e i n the binding of tRNA to ribosomes. The binding i s s p e c i f i c for 50S ribosomal subunits; 30S subunits do not have any a f f i n i t y for tRNA i n the absence of mRNA. Quantitative studies showed that there i s only one binding s i t e per 70S or 50S ribosome (148). According to Cannon et a l . (148) t h i s does not eliminate the p o s s i b i l i t y of several s i t e s , but i f there are, e i t h e r they are not a l l equivalent, or i f they are equivalent they have the property that binding tRNA to any one weakens the binding a b i l i t y of 16 the others. They suggest two models for the binding and exchange of tRNA. In the f i r s t the ribosome has one s i t e on which the tRNA binds i n rapid equilibrium with the tRNA i n s o l u t i o n . 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 dissociates very slowly. The exchange would take place by a second tRNA molecule a l t e r i n g the s i t e and d i s p l a c i n g the f i r s t . One can consider two equivalent s i t e s f o r the binding : e i t h e r s i t e alone can bind the tRNA t i g h t l y but i f both are f u l l , both tRNA molecules are bound loosely. Such a structure would show rapid exchange associated with the loose binding for the two molecules but only slow loss and t i g h t binding with one tRNA. The basic d i s t i n c t i o n between the two models l i e s i n the composition of the intermediate state during the exchange process : the intermediate state i n the second model i s a ribosome and two bound tRNA molecules, i n the f i r s t model i t i s a free r i b o -some. In the absence of protein synthesis or mRNA, and at 0.1 mM Mg + + ion concentration, the ribosomes are dissociated i n t o t h e i r subunits and i n the ensuing process a l l the bound tRNA i s completely removed (148). A f t e r protein synthesis in a c e l l - f r e e extract of c o l i , a small f r a c t i o n of the tRNA that i s bound to the ribosomes i n high magnesium (10 mM) becomes r e s i s t a n t to being washed of f the ribosomes i n low magnesium (0.1 mM). This amounts to about h a l f a molecule of tRNA per ribosome (148) . This has been interpreted as 17 due to the presence of a nascent polypeptide chain on the tRNA which stabilizes the binding of the tRNA to the ribosome. Takanami (252, 255) has also observed, using rat l i v e r ribosomes, that the ribosomes w i l l bind tRNA on a roughly one-for-one basis. This binding i s magnesium dependent, but after in vitro protein synthesis, some of the bound tRNA remains tightly bound to ribosomes in low magnesium. He has shown that this tightly bound tRNA is not covalently linked to the rRNA but rather is attached to the end of the nascent polypeptide chain. (b) Non-Enzymatic Specific Binding of Aminoacyl-tRNA In the presence of template RNA, tRNA attaches to ribo-somes with specificity (149) and inhibits the binding of aa-tRNA to ribosomes, presumably by competing with aa-tRNA for ribosomal binding sites (255). Poly U stimulates the Phe binding of both deacylated tRNA and Phe-tRNA to ribosomes (250). At high Mg T T ion concentration, tRNA binds to ribosomes with approximately the same a f f i n i t y , and to the same extent as Phe-tRNA. Since both tRNA and aa-tRNA recognize codons, the ratio of tRNA to aa-tRNA may sometimes regulate the rate of protein synthesis. I t has been shown, however, that with addition of a soluble enzyme fraction and GTP, the inhibition of Phe-tRNA binding to ribosomes by tRNA is 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 inhibition, due to tRNA, of AUG-dependent binding of N-fMet-18 tRNA. Specific binding could be shown to occur in solution of high Mg + + ion concentration; a binding which was enhanced by K* or NH i ,* ions (149-152) . This finding does not demand a special energy source other than the thermal energy of the reacting components. The in a b i l i t y of the mRNA-ribosome complex to distinguish 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 is not involved in the translation step (153-155) . With respect to the binding forces that mediate the mRNA-dependent binding of tRNA to ribosomes, this result, in addition, 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 hint as to the number of specific tRNA-binding sites on the ribosome came from the discovery that poly U-directed binding of phenylalanine tRNA to 30S ribosomal subunits i s stimulated approximately twofold by the present of 50S ribosomal subunits at high Mg + + ion concentration and in the absence of protein synthesis (156-158). This finding i s consistent with the hypothesis that one molecule of aa-tRNA can bind to the 30S particle (acceptor site) and that the second binding site is 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 interact nonspecifically with the ribo-some. I t i s tempting to assume that the 30S subunit of the 70S ribosome has this function. In fact, 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 to bind aa-tRNA i n response to mRNA indicates, besides the s p e c i f i c codon-anticodon r e l a t i o n s h i p , a strong nonspecific i n t e r a c t i o n between aa-tRNA and the 30S ribosomal subunit (156, 159). Furthermore, the response of the s p e c i f i c aa-tRNA binding reaction to a large array of i n h i b i t o r s of protein synthesis, some of which act s p e c i f i c a l l y on the 30S ribosomal subunit, provides some evidence for the nonspecific a s s o c i a t i o n between the ribosome and a part of the aa-tRNA molecule which comprises base sequences outside of the anticodon region. Yet, i t i s very l i k e l y that both ribosomal subunits of the 70S ribosome take part i n the nonspecific binding. The inte g r a t i o n of peptidyl-transferase within the structure of the 50S ribosomal subunit implies the occurrence of two tRNA-binding s i t e s on the 50S p a r t i c l e accessible to a l l tRNA molecules (160) . Further support comes from the observation that a n t i b i o t i c s which act s p e c i f i c a l l y on the 50S ribosomal subunit i n h i b i t the binding reaction (158) . Moreover, the aminoacyl ester bond of the aa-tRNA bound to a 70S ribosome i n response to mRNA i s protected from a l k a l i n e hydrolysis (161). Because the presence of the 50S p a r t i c l e i s absolutely necessary for the protection of the ester, the tRNA appears to be intimately associated with the 50S p a r t i c l e . The same conclusion was drawn from the observation that aa-tRNA bound to 70S ribosomes i n the presence of a template i s r e s i s t a n t to digestion by pancreatic RNase; again, the protection occurs 20 as a r e s u l t of the association of aa-tRNA with the 50S r i b o -somal subunit (162). For s p e c i f i c binding of tRNA to 30S ribosomal subunits, the adenosine terminus i s not important, but the binding to 70S ribosomes involves both the anticodon region and the terminal adenosine of tRNA (163) . In summary, the successful formation of a peptide bond requires the co-operative i n t e r a c t i o n between a l l components that p a r t i c i -pate i n t h i s reaction (164). Studies concerning the substrate s p e c i f i c i t y at the c a t a l y t i c center of the 50S ribosomal peptidyl transferase should make possible a more de t a i l e d c haracterization of tRNA-binding s i t e s on the ribosome. In general, the function of peptidyltransferase seems to be favored by s p e c i f i c i t y at the acceptor s i t e toward substrates with a free a-amino group and by the s p e c i f i c i t y at the donor s i t e toward substrates with an amido group i n that p o s i t i o n (165). There are almost no experimental data on the nature of these nonspecific binding forces. I t was found that treatment of 70S ribosomes with p r o t e o l y t i c enzymes under mild conditions abolished the binding capacity of ribosomes for s p e c i f i c tRNAs (166). Their capacity to bind mRNA was not affected by t h i s treatment. Although i t leaves the question unanswered whether ribosomal 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 i n the binding of tRNA to ribosomes, t h i s r e s u l t nevertheless provides evidence that the s p e c i f i c tRNA-binding reaction requires the s t r u c t u r a l i n t e g r i t y of the ribosome. Treatment of ribosomes 21 with p-dinitrofluorobenzene, that causes loss of the mRNA-directed tRNA binding a c t i v i t y , y i e l d s some weak i n d i c a t i o n that amino-, t h i o l - , phenol-, or imidazol-groups of ribosomal proteins are involved, d i r e c t l y or i n d i r e c t l y , i n the i n t e r -a ction of tRNA with the template-ribosome complex (167) . Furthermore, the reaction of ribosomes with d i e t h a n o l d i s u l f i d e destroyed the aa-tRNA binding a c t i v i t y , whereas the association of mRNA with ribosomes modified i n such a way proceeded normally (168). The loss of binding a c t i v i t y may be due to the f a c t that free thiol-groups are necessary to maintain the proper configuration of the ribosomal binding s i t e or that they p a r t i c i p a t e d i r e c t l y i n the binding reaction. Since the binding of N-acylated aa-tRNA or peptidyl-tRNA i s not affected by sulfhydryl-reagents but only the binding of aa-tRNA, i t was concluded, f i r s t l y , that the binding s i t e f or p e p t i d y l -tRNA i s d i s t i n c t from that for aa-tRNA and, secondly, that only the acceptor s i t e i s altered by su l f h y d r y l reagents. The 30S ribosomal subunit i s the major s i t e of i n a c t i v a t i o n (169). Sul f h y d r y l reagents do not i n h i b i t the formation of poly-phenylalanyl-puromycin or of formylmethionyl-puromycin catalysed by 50S ribosomal subunits (160). The occurrence of a pentanucleotide of constant sequence i n a single-stranded loop of the clover l e a f model of a l l tRNA-species analyzed so far and the existence of 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 ribosomal 5S RNA, mediated by hydrogen bonds (258-259). 22 (c) Enzyme-Specific Binding of Aminoacyl-tRNA This w i l l be discussed i n the following section t i t l e d "The Ribosome and Protein Synthesis (see pages 24-28 ). (d) Summary There are s i t e ( s ) on the ribosomes which bind tRNA. The actual number of s i t e s i s unknown and may depend on the experimental conditions. For instance, Warner and Rich (260) using 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 conclusion that each ribosome active i n protein synthesis has two molecules of tRNA attached to i t . By contrast, the i n a c t i v e s i n g l e ribosomes bound approximately one molecule of tRNA, and t h i s binding was less firm than that seen i n the polysomes. In t h e i r extensive study of i n v i t r o binding of tRNA to c o l i ribosomes, Cannon et al.(148) found that washed ribosomes bound one molecule of tRNA per ribosome and t h i s was r a p i d l y exchangeable at 4°. Under conditions of a l l - f r e e protein synthesis, the same amount of tRNA became attached to the ribosomes, and a portion of t h i s was more firmly bound. On the other hand, Takanami (252, 261) had found that tRNA w i l l become attached to r a t l i v e r ribosomes only i n the presence of a transfer enzyme during incubation. With polysomal r e t i c u l o c y t e ribosomes there i s l i t t l e or no exchange at 4°, while i n a c t i v e ribosomes have a l i m i t e d exchange (260). I t i s possible that some of these differences are due to the various species involved. The i n v i t r o environment d i f f e r s i n many respects from the environment i n vivo. In p a r t i c u l a r , 23 the rate of protein synthesis i s so much lower i n v i t r o that i t i s not c l e a r that the difference between the amount of tRNA bound by active and inactive ribosomes would have been detected i n the i n v i t r o studies. According to Wettstein and N o l l (262) ribosomes engaged i n p r o t e i n synthesis bind at l e a s t two and at most three tRNA molecules and that the tRNA bound to r a t l i v e r polyribosomes occurs i n three d i f f e r e n t states which e x h i b i t d i f f e r e n t binding properties. Both the free aminoacyl-changed and the peptide-linked tRNA are t i g h t l y bound and not removable by washing, even at 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 of the active complex i s preserved. Moreover, t h e i r attachment i s i r r e v e r s i b l e and requires transfer enzymes as we l l as energy. These three d i f f e r e n t states i n which ribosome-bound tRNA i s encountered correspond to at l e a s t two, and probably three, d i s t i n c t s i t e s on the active 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. Single ribosomes devoid of mRNA do not have t h i s s e l e c t i v e binding s i t e . The second s i t e , condensing s i t e , which i s found on the 50S ribosomal subunit can bind tRNA i n the absence of mRNA; however, during protein synthesis t h i s s i t e i s only accessible from the activated state of the f i r s t s i t e . G i l b e r t has shown that tRNA-linked nascent poly-phenylalanine remains attached to 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 of the ribosomes into subunits (263) . Elson (264-265) , on the other hand, observed the release of 24 4S RNA from the 50S p a r t i c l e during i t s conversion to a p a r t i c l e with a lower sedimentation c o e f f i c i e n t i n the presence of high s a l t . The t h i r d s i t e , e x i t s i t e , i s s p e c i f i c for uncharged tRNA. Not a l l of the three s i t e s are equally occupied during protein synthesis i n v i t r o . During protein 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 conditions, a l l of the condensing and e x i t s i t e s , but somewhat less than h a l f of the decoding s i t e s are f i l l e d at any one moment. This would indicate that the 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 of tRNA, and i n vivo i t i s the formation of the peptide bond. The Ribosome and Protein Synthesis Although ribosomes may have several other functions i n vivo, for example, stimulation of RNA synthesis, or regulation of the biosynthesis of RNA or of ribosomes themselves, t h e i r only c l e a r l y established functions are those related to the synthesis of proteins (70) and th i s i s discussed below. In prokaryotes the i n i t i a t i o n of protein synthesis requires the formation of an i n i t i a t i o n complex consisting of the 30S ribosomal subunit, mRNA and formyl methionyl-tRNA (fMet-tRNA^) (9-11). In a so l u t i o n of low Mg + + ion concentra-t i o n i n i t i a t i o n factors as w e l l as GTP are required for t h i s step. I n i t i a t i o n factors are proteins which were o r i g i n a l l y obtained from crude ribosomes by washing with IM NH^Cl and were found to be required for the t r a n s l a t i o n of natural mRNA (12-14). At l e a s t three i n i t i a t i o n f a ctors, F i , F 2 and F 3 25 (also c a l l e d A, B and C respectively) are known (15. 16). The p o s s i b i l i t y of the presence of a new i n i t i a t i o n f a c t o r , i s now undergoing i n v e s t i g a t i o n . In vivo the i n i t i a t i o n s i t e on the natural mRNA contains an AUG codon which codes for fMet-tRNA f (17-19). The codons AUG and GUG serve as i n i t i a t o r codons i n v i t r o . The presence of a tRNA involved s o l e l y i n the i n i t i a t i o n of pro t e i n synthesis i n b a c t e r i a l systems was discovered by Marcker and Sanger (71). This tRNA so f a r appears to i n i t i a t e synthesis of a l l b a c t e r i a l proteins (72-75) and probably a l l proteins i n mitochondria (76, 77), chloroplasts (78) and blue-green algae (79). Preliminary experiments indicate that i n some mammalian systems the mRNA codon assignments f o r peptide chain i n i t i a t i o n are i d e n t i c a l to b a c t e r i a l systems (235-239, 245) . c o l i has two methio-Met Met nine accepting tRNAs : tRNA* and tRNA . These are both t m changed by the same methionyl-tRNA synthetase (80) but only Met methionine attached to tRNA^ can be formylated by a trans-formylase (17) which has been p u r i f i e d from c o l i (81). Very recently transformylases from wheat germ chloroplasts (229) and from Saccharomyces cerevisae mitochondria (230) have been i s o l a t e d . According to Ochoa (117-118), the forma-t i o n of the i n i t i a t i o n complex involves two steps : (a) the F i and F3-dependent binding of natural mRNA to the ribosomes, and (b) the GTP requiring F 2-dependent binding of fMet-tRNA^ to the mRNA-ribosome complex. A f t e r formation of the i n i t i a t i o n complex co n s i s t i n g of the 30S ribosomal 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 ribosomal subunit joins to form the 70S i n i t i a t i o n complex (23, 24). Although the d e t a i l s are s t i l l unclear, studies indicate that fMet-tRNA^ i s i n i t i a l l y attached to the A s i t e (on the 30S subunit) and i s subsequently translocated to the P s i t e (on the 50S subunit). GTP i s cleaved before or during t h i s t r a n s l o c a t i o n (25, 82). The next step i s the binding of a second aminoacyl-tRNA to the A s i t e . This binding i s directed by the codon next to AUG and requires GTP as well as two soluble protein f a c t o r s , Ts and Tu (26). The T factors (Ts and Tu) i n t e r a c t f i r s t with GTP and then with an aminoacyl-tRNA, with the exception of fMet-tRNA f and Met-tRNAf (27). The GTP-aminoacyl-tRNA-T factor complex then reacts with the ribosome leading to binding of the aminoacyl-tRNA at the A s i t e . GTP appears to be hydrolyzed at t h i s step (28). The next step i n protein synthesis i s the formation of a peptide bond between fMet-tRNA^ (or peptidyl-tRNA) and the second aminoacyl-tRNA bound to the ribosome. This peptide bond formation does not require any supernatant protein fa c t o r , and i s catalyzed by peptidyl transferase, an enzyme present on the 50S ribosomal subunit (30) . The peptide bond formation occurs by transfer of the acyl group at the P s i t e to the amino group of the aminoacyl-tRNA at the A s i t e . The energy for peptide bond formation i s supplied by the r e l a t i v e l y high energy ester bond between the tRNA and the peptidyl moiety (83). A f t e r formation of the f i r s t dipeptide bond, the fMet 27 aminoacyl-tRNA stays at the A s i t e , and the discharged tRNA^ stays on the ribosome, probably at the o r i g i n a l P s i t e . The next step, t r a n s l o c a t i o n , involves movement of fMet aminoacyl-tRNA (or peptidy1-tRNA) from the A s i t e to the P s i t e . Release of discharged tRNA^ from the P s i t e accompanies t h i s t r a n s l o c a t i o n step (31). Translocation requires a soluble p r o t e i n factor (G factor) and GTP which i s hydrolyzed to GDP and P i (32). Factors s i m i l a r to T and G and which function i n the same manner have been i s o l a t e d from mammalian systems (238-242) and yeast (243) . Simultaneously with the trans l o c a t i o n , the ribosome moves along the mRNA, i n the 5'-to 3'-direction, by the length of one codon, leaving the t h i r d codon ready for the binding of a new aminoacyl-tRNA to the A s i t e . These processes are repeated and polypeptide chain elongation continues u n t i l the ribosome encounters one of the chain termination codons (UAG, UAA and UGA). Recently the mRNA codon assignments for peptide chain termination have been found to be i d e n t i c a l i n mammalian systems (97, 233). For termination to take place, the peptidyl-tRNA must be on the P s i t e (37, 38). Chain termination leads to cleavage of the polypeptide from the tRNA and the subsequent release of th i s tRNA from the ribosome. Since i n i t i a t i o n of protein synthesis takes place on the 30S ribosomal subunit, the ribosomes a f t e r protein synthesis must subsequently undergo d i s s o c i a t i o n . Subramanian et a l . (110) i s o l a t e d a d i s s o c i a t i o n factor (DF) from the 30S f r a c t i o n f r a c t i o n which was found to carry out t h i s function. Very 28 recently i t has been shown (111-112, 231) that t h i s DF factor was a c t u a l l y i n i t i a t i o n factor F 3 . Therefore F 3 has now been shown to have both RNA binding and ribosome d i s s o c i a t i o n a c t i v i t i e s . Some workers believe that chain termination l i b e r a t e s 70S ribosomes (113-114) which d i s s o c i a t e subsequently (113) by i n t e r a c t i o n with F 3 , released from 30S ribosomal subunits when the 50S ribosomal subunit joins the 30S i n i t i a -t i o n complex. This would imply that F 3 recycles between a ribosome-bound and a free form. Other investigators hold the view that ribosomes d i s s o c i a t e at chain termination (115-116, 120). I f so, F3 might remain ribosome-bound throughout the e n t i r e cycle so long as ribosomes, bearing F 3 , can associate and remain associated when they carry aminoacyl- or p e p t i d y l -tRNA. At present, the precise manner i n which F 3 functions i n the ribosome cycle remains an open question. Release Factors and the Mechanism of Termination When a ribosomal : peptidyl-tRNA complex reaches a terminator codon on a mRNA, some mechanism must bring about hydrolysis of the ester bond between the peptidyl and tRNA moieties. This would then allow the completed protein to be released. Most of the information about the mechanism of chain termination was obtained i n two assay systems. In one of these, RNA from a mutant R17 or f 2 phage with a UAG nonsense codon early i n the coat protein gene was used as the messenger. In v i t r o , t h i s messenger d i r e c t s the synthesis of the free 29 (not tRNA-linked) amino terminal hexapeptide of the coat protein (75, 84-85). Bretscher (84) prepared a c e l l - f r e e system which included only those aa-tRNA species needed for forming the hexapeptide. I f a codon i s untranslatable i n consequence of the lack of a required aa-tRNA, t h i s leads to the stoppage of peptide chain propagation but not to chain termination. The f a c t that, i n the presence of the mRNA from a nonsense mutant, chain termination occurred even i n the absence of a l l tRNAs, except the six species added, suggested that i f RNA was involved i n chain termination, i t was not contained i n the tRNA f r a c t i o n (75, 84, 86-87) obtained from the high speed supernatant from c o l i . Capecchi (75) was able to prepare a substrate for studying the mechanism of termination i n the following way. The formation of a hexa-peptidyl-tRNA as s p e c i f i e d by the R17 RNA was blocked at the pentapeptidyl-tRNA stage by omitting from the i n v i t r o system the amino acid coded by codon s i x . The ribosome-mRNA-penta-peptidyl-tRNA complex was then separated from the supernatant f r a c t i o n by ce n t r i f u g a t i o n and the aa-tRNA needed to complete the hexapeptidyl-tRNA was added i n the presence of GTP. The l a s t amino a c i d was then added to the coat protein fragment. The r e s u l t i n g hexapeptidyl-tRNA remained attached to the mRNA-ribosome complex. This product made possible the study of the unique requirements of the release step. I t was found that the release of free hexapeptide from th i s complex depended on a protein component from the high speed supernatant of c o l i This component was designated release factor (R factor) (43). 30 Following Capecchi's lead, Nirenberg's group has been very active i n studying the factors involved i n chain termina-t i o n (88-98) . Caskey et a l . (88) used a d i f f e r e n t termination assay. fMet-tRNA^ was bound to ribosomes i n the presence of the t r i n u c l e o t i d e AUG to form an AUG-fMet-tRNA^-ribosome complex. Then a terminator t r i n u c l e o t i d e and crude R factor were added and the release of free fMet was measured. With t h i s assay they were able to resolve R into 2 components, Rx and R 2 ; Ri responded to UAG and UAA but not UGA whereas Rj was active with UGA and UAA but not UAG (89, 91). Capecchi and K l e i n (107) used antisera to p u r i f i e d Rx and R 2 t o . t e s t t h e i r r o l e i n release of completed proteins i n a c e l l - f r e e system dir e c t e d by R17 RNA. Their r e s u l t s indicated that these factors were required for release of completed proteins and that e i t h e r factor could promote release of ei t h e r the coat protein or the r e p l i c a s e ; t h i s implied that both c i s t r o n s terminated with UAA since t h i s was the only one of the three terminator codons recognized by both Ri and R 2 . Nichols (108) sequenced the portion of R17 RNA at the end of the coat protein c i s t r o n and found two consecutive terminator codons, UAAUAG. This may mean that, at l e a s t i n some systems, two terminators are required to ensure that release occurred between c i s t r o n s . This seems most l i k e l y i n view of the existence of nonsense suppressors which allow UGA or UAG to be read as specifying an amino a c i d , instead of serving as terminator s i g n a l s . A t h i r d component, S, has been found to increase the rate of formation or s t a b i l i t y of the terminator codon : ribosome : 31 factor R complex (90, 92). Peptidyl transferase may also be involved i n release since i n h i b i t o r s of this enzyme also i n h i b i t release. The presence of the terminator complex may allow peptidyl transferase to act as a hydrolase and thus break the ester bond holding the completed protein to the l a s t tRNA (peptidyl-tRNA) (93, 99). I t was previously suggested that the non-ribosomal enzyme, N-acylaminoacyl-tRNA hydrolase may catalyze the release reaction (102, 106) but t h i s now seems u n l i k e l y i n view of the results of Caskey et a l . (88) who showed that fMet-tRNA i s cleaved during the release reaction while i t i s known that free fMet-tRNA i s a very poor substrate f o r the hydrolase. Recently Jost and Bock (232) i s o l a t e d an N-substituted aminoacy1-tRNA hydrolase from yeast supernatant. They suggested that there are two hydrolases, one, a ribosomal hydrolase, may be involved i n the release of nascent peptide chains during the normal process of chain termination, and the other, the supernatant hydrolase, could hydrolyze any oligopeptidy1-tRNA present i n the c e l l sap. Recent studies indicate that R f a c t o r and suppressor tRNA compete for the t r a n s l a t i o n of terminator codons (96, 100). Recently Goldstein et a l . (97, 233) have i s o l a t e d an R factor from rabbit r e t i c u l o c y t e s which, i n the presence of the fMet-tRNA-AUG-ribosome complex and the UAA terminator codon causes the release of fMet. They also found that a n t i b i o t i c s which i n h i b i t e d peptidyl transferase a c t i v i t y also i n h i b i t e d release. Very recently Ishitsuka and K a j i (109) i s o l a t e d another 32 factor c a l l e d tRNA release factor (TR) from the high speed supernatant of c o l i . I t was found to f a c i l i t a t e the removal of tRNA from ribosomes and was d i s t i n c t l y d i f f e r e n t from the G factor i n that i t did not require GTP for i t s action. They postulate that t h i s factor may act at the l a s t step i n protein synthesis i n the following way : A completed polypeptide chain linked to tRNA through the -COOH terminal group of the polypeptide i s probably bound to the donor s i t e of the ribosome with the acceptor s i t e having the termination codon. The codon-specific chain-termination factor would then bind to the acceptor s i t e of the ribosome r e s u l t i n g i n the s p l i t t i n g of the polypeptide group from the tRNA. The tRNA i s l e f t on the donor s i t e of the ribosome and cannot be removed by the G factor which releases tRNA only as a conse-quence of t r a n s l o c a t i o n . The TR factor would then remove the l a s t tRNA from the ribosome. This hypothesis complements that suggested by Vogel e t a l . (104) who postulated that peptidyl transferase might cleave the substrate, with the R factor acting to change the s p e c i f i c i t y of the peptidyl transferase reaction, so that nascent peptide was now trans-ferred to water instead of to a molecule of aa-tRNA. In t h i s event, the R factor might i n t e r a c t d i r e c t l y with peptidyl transferase, i n which case the factor could be looked on as part of a multimeric release enzyme. A l t e r n a t i v e l y , the R factor might i t s e l f cleave the bond between nascent peptide and tRNA. The factor has been shown to be unable to do t h i s 33 when free in solution (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 reaction—the 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 the termination signals directly or whether other molecules, which in turn interact with the appropriate species of R factors, are involved in this process. Ribosomal Structure and Function The foregoing discussion had illustrated the complexity of ribosome structure and some of the known events of i t s function in protein biosynthesis. Related to this i s the recent finding by Lodish (212) that ribosomes from c o l i i n i t i a t e d synthesis in vitro of a l l three of the proteins coded by phage f 2 , but ribosomes from stearothermophilus i n i t i a t e d synthesis of only one. He was able to show that 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 : the origin of the 50S ribosomal subunits, i n i t i a t i o n factors, tRNA or supernatant enzymes had no effect. Thus the 30S particle 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 translation i s dependent on the conformation of mRNA (244) . Presumably the tertiary structure of the ribosome plays a crucial role. The work of Steitz (18) 34 and Hindley (19) suggested that the s i t e on the messenger to which the 30S p a r t i c l e attached was i d e n t i c a l to the s i t e at which protein synthesis was i n i t i a t e d . I t has also been shown that i n f 2 RNA there are AUG sequences which can i n i t i a t e p rotein synthesis, but are prevented by the RNA structure from doing so (18, 19, 213-215) . To make the problem even more complicated, a f t e r i n f e c t i o n of c o l i by phage ribosomes bind appreciably only to the maturation protein i n i t i a t i o n s i t e of R17 RNA (216) . Apparently t h i s change l i e s i n the i n i t i a t o r factor f r a c t i o n . Recently i n order to get a cle a r e r p icture of the ribosome, studies on the low molecular weight RNA bound to ribosomes have been started by many groups with the hope of being able to c o r r e l a t e the findings with a possible c l e a r e r understanding of the structure of the ribosome with respect to i t s function. U n t i l recently RNA was considered to belong to one of those categories : tr a n s f e r , ribosomal or messenger. Develop-ment of more sophisticated a n a l y t i c a l techniques, such as polyacrylamide gel electrophoresis and improved chromatographic techniques, has permitted the detection of a number of new RNA species, among which are RNAs of r e l a t i v e l y low molecular weight (5-8S) . Such small RNAs have been found i n or associated with ribosomes (170, 175), microsomal membrane (185), smooth endoplasmic reticulum (186), n u c l e i (181, 182, 187, 188, 191, 194) and n u c l e o l i (188, 190, 193). Knight and Sugiyama (170) found a new class of tRNA i n 35 E. c o l i ribosomes and supernatant and i n HeLa c e l l mitochondria but not i n HeLa c e l l cytoplasm. Most of the tRNA i s not associated with ribosomes. This minor tRNA was found to have a molecular weight, judged by mobility on polyacrylamide gels, intermediate between the major part of tRNA and 5S RNA. The minor tRNA of E^ c o l i can be changed with amino acids, however, i t s amino acid s p e c i f i c i t y i s d i f f e r e n t from that of the major class of tRNA. A further study by Knight (171) revealed that ethidium bromide treatment of growing HeLa c e l l s eliminated t h i s minor tRNA from the mitochondria and reduced the major species by 50% without a f f e c t i n g the synthesis of 5S RNA. The minor species was found to be unmethylated while the major species had a completely d i f f e r e n t methylation pattern from the cytoplasmic 4S RNA. No s i g n i f i c a n t differences were observed between the 5S RNA of the cytoplasm and that occurring i n the mitochondrial f r a c t i o n . Recently precursor tRNA (pre-tRNA) has been i s o l a t e d from HeLa c e l l cytoplasm (183, 184) . This pre-tRNA which i s not methylated migrated on gel electrophoresis between tRNA and 5S RNA. I t has also been shown that the tRNA from the mitochondria of r a t l i v e r and Neurospora have 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 cytoplasmic counterparts (172, 173). The mitochondrial tRNA was found to hybridize more e f f i c i e n t l y to the mitochondrial DNA than the corresponding cytoplasmic tRNA suggesting that mitochondrial tRNA i s transcribed from mitochondrial DNA. In a recent report, Nass and Buck (174) observed that the four mitochondrial tRNAs 36 they studied from r a t l i v e r d i f f e r e d i n base sequences from t h e i r cytoplasmic counterparts. These mitochondrial tRNAs could only be acylated by mitochondrial synthetases and not by cytoplasmic enzymes. Recently 7S RNA has been i s o l a t e d from HeLa c e l l r i b o -somes (175). I t was found to be associated with the 28S rRNA and as such may play a r o l e i n the conformation of the 28S rRNA. They also present evidence that 7S RNA derives from the same polynucleotide precursor as does i t s accompanying 28S molecule (175, 217) . 7S RNA was also found i n Chinese hamster and chicken f i b r o b l a s t 28S rRNA (175). There appears to be one 7S RNA molecule for every 28S molecule and i t appears to be attached to the larger rRNA by non-covalent bonds. The 7S contains approximately one methylated base. Electrophoretic mobility of 4, 5 and 7S RNA reveals about an equal separation between 4 and 5S RNA and 5 and 7S RNA. Weinberg and Penman (181) have observed s i x d i s t i n c t low molecular weight species of RNA i n the nucleoplasm and nucleolus of HeLa c e l l s . The nuclear RNA species range i n s i z e from 4 to 6S RNA, and appear to be stably associated with the nucleus. Some are associated p r i n c i p a l l y with the nucleoplasmic and others with the nucleolar f r a c t i o n . These species are methylated and have base compositions unlike any of the other nuclear RNA classes. These species represent only about 0.4% of the t o t a l c e l l u l a r RNA. In the nucleus, 5S RNA i s present i n great excess r e l a t i v e to 7S RNA (175; 37 th i s RNA exhibits both the sedimentation v e l o c i t y and e l e c t r o -phoretic mobility of a species of approximately 5.5S RNA, the term 7S RNA o r i g i n a l l y given to t h i s species by Pere et a l . i s thus a misnomer) which i s consistent with the existence of a sizeable pool of 5S ribosomal RNA i n the nucleus. The 5.5S and 5S RNA species are present i n equimolar quantities i n cytoplasmic ribosomes. Similar species of nuclear RNA are found i n mouse f i b r o b l a s t cultures and i n the developing chick embryo br a i n . Preparative gel electrophoresis has been used to i s o l a t e four methylated low molecular weight RNA molecules from Chinese hamster c e l l s (187). They have been shown to be d i s t i n c t from tRNA, 5S and 5.5S rRNA and are found i n a f r a c t i o n enriched with respect to n u c l e i . In a more recent 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 three new nuclear low molecular weight RNA species (182) . One species migrates with i d e n t i c a l electrophoretic mobility to that of 5S rRNA and i s methylated while the other two species, also methylated, migrate slower than 5.5S RNA. These RNAs are found almost s o l e l y i n the nu c l e i of interphase c e l l s and are quite loosely associated with the nucleoprotein complexes of the nucleus which i s i n marked contrast to the other previously described s i x species of nuclear RNA. A l l nine nuclear RNA species d i f f e r from each other i n several of t h e i r properties; for example, s t a b i l i t y , s i z e , synthesis, quantity, etc., which may r e f l e c t differences i n t h e i r function i n vivo. Busch's group has shown that the nuclei and n u c l e o l i of 38 normal r a t l i v e r (188) and a number of tumor c e l l s (189) consistently contained 4 to 6S RNA. Recently nucleolar 4 to 6S RNA has been i s o l a t e d from Novikoff hepatoma asc i t e s c e l l s and separated into three main fr a c t i o n s by exclusion chromato-graphy on Sephadex G-100 (190). The e l u t i o n pattern was found to be s i m i l a r to the ribosomal 4 to 6S RNA of the Novikoff hepatoma. Peak I (the f r a c t i o n that emerged i n the void volume) containd two major components, both of lower mobility than ribosomal 5S RNA. Only one band migrated i n 10% poly-acrylamide gels and was s i m i l a r to the RNA derived from ribosomes. The main component i n Peaks II and III migrated with m o b i l i t i e s s i m i l a r to 5S and 4S RNA r e s p e c t i v e l y . Peacock and Dingman (191) reported the presence of several low molecular weight RNA species (4 to 7S RNA) i n r a t l i v e r n u c l e i which were not present i n the cytoplasm. Hodett and Busch i s o l a t e d t h i s nuclear f r a c t i o n and characterized two U-rich RNA f r a c -tions (192, 195, 197). These U-rich f r a c t i o n s , with mobility less than 5S RNA, are apparently unrelated to those found by other workers for HeLa c e l l ribosomes (175) and cytoplasmic f r a c t i o n s (185). Each of these other fract i o n s seem to be present p r i m a r i l y i n the rRNA or ribosomal subfractions. Busch suggests that since t h i s U-rich RNA has a lower rate of l a b e l i n g than any other nuclear RNA, t h i s RNA would appear to be stable and might serve a s t r u c t u r a l r o l e but since this RNA i s less hydrogen-bonded than tRNA or rRNA i t might also exert some role i n template a c t i v i t y . Recently two other low mole-cular weight RNAs have been l i b e r a t e d from the nucleolar 28S 39 RNA f r a c t i o n and referred to as 8S and U3 RNA (193) . These two RNAs are not associated with ribosomal 28S RNA. The molar r a t i o of 8S and U3 RNA to nucleolar 28S RNA i s only approximately 1:2, suggesting that only some of the nucleolar 28S RNAs are bound to these molecules. They may have some ro l e i n the formation of the ultimate cytoplasmic ribosomal p a r t i c l e . I t i s possible that they may also serve as e s s e n t i a l components for the movement of nucleolar products to the nuclear ribonucleoprotein network, or for the addition of sp e c i a l proteins that are components of the ribosomes. An 8S nuclear RNA has also been i s o l a t e d from KB c e l l s (246) . More recently U3 RNA and 4.5S RNA have been i s o l a t e d from the nucleus of Novikoff hepatoma c e l l s and from r a t l i v e r n u c lei (194-195). This 4.5S RNA was subsequently separated into three f r a c t i o n s . I t should be noted that the 3'-terminal of one f r a c t i o n i s blocked (196). Recently the nucleolar U3 RNA was separated into 4 d i s t i n c t bands (198) . I t has been suggested that these RNAs may function i n processing of nucleolar ribosomal RNA precursors into ribosomal precursor p a r t i c l e s and f i n a l l y into mature ribosomes. The low turnover of these molecules suggests that some might be stable messenger RNAs for c e r t a i n proteins, perhaps ribosomal or ribosomal precursor proteins. I t i s also possible that these RNAs may play a ro l e as i n i t i a t i o n factors i n ribosomal RNA synthesis. Various RNAs have been i s o l a t e d from rabbit r e t i c u l o c y t e s besides 4, 5 and 7S RNA (207-208). Two RNAs sedimenting at 8S and 10S have properties expected of mRNA. 40 Sea urchin 26S rRNA has been shown to contain a hydrogen-bonded 5.8S rRNA (199), which i s unmethylated and which i s s i m i l a r to the 5.5S RNA associated with rRNA of HeLa c e l l s (175) , chicken f i b r o b l a s t s (175) and Novikoff ascites tumors (193) . Sy and McCarty suggest (199) that the 5.8S rRNA or the 5.5S rRNA i s involved i n maintaining the correct 3-dimensional configuration of e i t h e r the 28S or 26S rRNA necessary for i t s proper i n t e r a c t i o n with proteins i n ribosome maturation. Yeast ribosomes have been found to contain besides 4S and 5S RNA, a 5.8S RNA molecule which i s unmethylated and which i s non-covalently attached to the 25S rRNA (200) . I t i s suggested that the 5.8S RNA i s derived from a part of the 35S precursor RNA, whereas the 5S RNA i s made de novo. I t has been found, j u s t recently, that the mitochondrial ribosomes of Neurospora crassa are devoid of 5S RNA (201) . A 7S RNA component was also absent. A f t e r i n f e c t i o n with adenovirus 2 or 7, human e p i t h e l i o i d c e l l s synthesize a d i s c r e t e species of low molecular weight RNA (176). This i s found predominantly i n the soluble f r a c t i o n of the cytoplasm of these c e l l s . I t s function i s unknown, and i t s primary structure i s d i f f e r e n t from transfer RNA and from the two p r i n c i p a l low molecular weight RNA components found i n the ribosomes of the uninfected KB c e l l s (177). Low molecular weight RNA synthesis i n T5-infected c o l i occurred predominantly 3 to 4 mins. a f t e r complete i n f e c t i o n (178). This type of RNA synthesis was characterized by seven d i s c r e t e bands of molecular weight range 8.0 to 3.1 x 10"* and a broad 41 band migrating equivalent to host 4S RNA. The data suggested that a l l species of molecular weight between 5.3 and 2.6 x 10 k were probably cleavage products of RNA of higher molecular weight. Only one band, molecular weight, 5.3 x 10" has been shown to be bound to polysomes. Altman has i s o l a t e d tRNA precursors from c o l i infected with bacteriophage <J>80 (205) . The precursor migrated on polyacrylamide gels between 4S and 5S RNA. Many species of low molecular weight RNA ranging from 4S to 10S have been i s o l a t e d from Rous Sarcoma Virus (179, 180). P u r i f i e d RSV contain a homogeneous population of methylated 4S RNA which i s indistinguishable from host tRNA on the basis of electrophoretic mobility, although differences i n nucleotide composition are detectable. A minor homogeneous RNA component, with sedimentation v e l o c i t y and electrophoretic mobility approximating the 7S RNA molecule, and a few methyl residues has been detected. I t s s i g n i f i c a n c e and possible function are presently obscure. Recently the nucleotide sequence of 6S RNA from the supernatant f r a c t i o n of c o l i has been established (202) . This RNA had been noted previously although no function has been assigned to i t (203-204) . I t s re l a t i o n s h i p with RNAs found i n higher organisms i s unclear. I t may be related to one or other of several bands of rather s i m i l a r electrophoretic m o b i l i t i e s to 6S RNA on acrylamide gel electrophoresis (believed to be nuclear RNAs) (191, 197). I t i s , however, u n l i k e l y to be related to the low molecular weight ribosomal 42 RNA, c a l l e d 7S RNA (175) , for 6S RNA of c o l i i s not found on ribosomes. In summary, a new and e x c i t i n g chapter has recently opened up i n the f i e l d of RNA chemistry and p a r t i c u l a r l y that of the biochemistry of nucleolar, nuclear and rRNA; for example, the uniquely l o c a l i z e d low molecular weight RNA. The eventual e l u c i d a t i o n of t h e i r functions w i l l help i n the understanding of ribosomal structure and function. 43 OUTLINE OF THE PROBLEM The events involved i n the i n i t i a t i o n of protein synthe-s i s are r e l a t i v e l y well understood. However, the mechanism of chain termination i n protein biosynthesis i s s t i l l an unsolved problem. The discovery of R factors was a great step forward, but i n contrast to the case of 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 involved, the requirement for a s p e c i f i c tRNA i n termination has not, as yet, been shown and the present understanding of the problem i s that such a tRNA i s not required. The chain-terminating experiments outlined i n a previous section (Release Factors and the Mechanism of Termina-t i o n , p. 2 8 ) i n which highly p u r i f i e d tRNAs were used, nevertheless were not properly c o n t r o l l e d since i t can be argued that the terminating tRNA could remain bound to the ribosomes. The previous investigators f a i l e d to show that t h e i r ribosomes or ribosomal subunits were devoid of 4S RNA. Perhaps t h i s hypothetical chain-terminating tRNA i s d i f f e r e n t i n some manner from the normal tRNA such that i t i s not removed from the ribosome during the cleaning procedure. In any event, i t may combine i n some manner with R factor, or some other protein, such as peptidyl transferase i n the termination mechanism, or i t may simply be present to s t a b i l i z e ribosomal conformation for the termination process. These are only some of the questions that must be answered before a f i n a l mechanism can be hypothesized. Recently investigations have led to considerable under-standing of the ribosomal structure. Both subunits have been reconstituted with p a r t i c u l a r emphasis on the 30S subunits and the order i n which the proteins are reassembled to form the f unctional u n i t . Less i s known about the b i o l o g i c a l r o l e of the RNA associated with ribosomes. In p a r t i c u l a r the nature and function of many small molecular weight RNAs associated with ribosomes i s unknown. Thesis Proposal This thesis was devoted to the i n v e s t i g a t i o n and charac-t e r i z a t i o n of the low molecular weight RNA bound to c o l i ribosomes which had been prepared by standard procedures. Methods were also studied for preparing ribosomes devoid of a l l 4S components with p a r t i c u l a r emphasis on the r o l e of tRNA i n the chain termination mechanism. Ribosomes from Ej_ c o l i were chosen for study not only because they are so well characterized and have been used i n previous chain termination studies but also because of the ease with which these active ribosomes can be obtained. The experimental o u t l i n e i s : (a) i s o l a t i o n and preparation of active ribosomes (WRIb), (b) removal of bound tRNA from WRib and subsequent ch a r a c t e r i z a t i o n , (c) characterization of the low molecular weight RNA from whole ribosomes and the subunits, and (d) exchange of labeled ribosomal bound tRNA with unlabeled tRNA and subsequent c h a r a c t e r i z a t i o n . 45 MATERIALS AND METHODS Chemicals Common chemicals obtained commercially were of the highest purity or reagent grade. Individual radioactive amino acids including the amino acid mixture ( 1 **C-labelled) and 3 H - l a b e l l e d u r a c i l were obtained from New England Nuclear Corporation; adenosine 5'-triphosphate (disodium) (ATP) and guanosirie 5'-triphosphate (trisodium) (GTP) , from Calbiochem; p o l y u r i d y l i c acid (poly U), from Miles Chemical Company; puromycin dihydrochloride (PM.2HC1), from N u t r i t i o n a l Biochem-i c a l s Corporation; N,N 1-Methylenebisacrylamide ( B i s ) , N,N,N',-N'-Tetramethylethylenediamine (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 acid (ATA), from Eastman Organic Chemicals; methylene blue from Fisher S c i e n t i f i c Co.; acridine orange (basic orange 14), acrylamide and 3'-dimethyl-aminopropionitrile (DMAPN), from Matheson, Coleman and B e l l ; lanthanum acetate from K&K Laboratories, Inc.; cesium chloride (CsCl), A grade for density gradients, from Calbio-chem; sucrose, density-gradient grade (ribonuclease-free), from Mann Research Laboratories, Inc.; transfer RNA (tRNA) from c o l i B, from General Biochemicals; and DNase I from Worthington Biochemical Corp. Preparation of E. c o l i B (a) C e l l s grown to the late log phase were obtained from the Grain Processing Co., Muscatine, Iowa. The growth medium used was as follows : 1% glucose, 1% yeast extract i n a 46 phosphate buffer medium, the buffers being monopotassium and dipotassium phosphate. The s t a r t i n g pH was 7.0 to 7.1. A f t e r the growth period was completed, the c e l l s were harvested as follows : (1) the contents of the fermentor were cooled from 37° to 16° and then centrifuged, (2) the c e l l s were recovered from the centrifuge and washed i n a medium made up of 0.5% KC1 and 0.5% NaCl, (3) the c e l l s were recentrifuged and frozen at -20°. (b) One hundred mis of medium (see below) i n a 125-ml e r l e n -meyer f l a s k was inoculated with c o l i previously grown on the surface of an agar s l a n t and the f l a s k was aerated by shaking at 100 rpm on a Metabolite Water Bath Shaker (New Brunswick S c i e n t i f i c Co., Inc.) set at 37°. When growth had reached mid-log phase, the contents of the f l a s k , which served as the inoculum, were added to a carboy containing 15 l i t e r s of the following minimal medium at pH 7.0 (g/L) : Throughout the growth period the c e l l s were vigorously aerated. When the c e l l s had reached the la t e log phase, they were quickly cooled by adding i c e and then harvested by cent r i f u g a t i o n and stored at -70°. Na citrate.2H 20 MgSO*.7H20 NIUC1 glucose K2HPO KH2POi, 7.0 3.0 0.5 0.2 1.0 4.0 autoclaved together for 20 mins. Preparation of E. c o l i B Pyrimidine-Requiring Mutant 12632 (American Type Culture C o l l e c t i o n , ATCC 13135) 47 CHART .1* Preparation of E. c o l i ribosomes c e l l u l a r debris E._ c o l i a) ground with glass beads i n buffer A (10 mMMg(0Ac) 2) b) centrifuged at 10,000 x g supernatant a) incubated with DNase b) centrifuged 2x at 30,000 x g p r e c i p i t a t e 1 supernatant centrifuged 105,000 x g at ribosomal p e l l e t supernatant a) mixed with (S100) buffer B (0.1 mM Mg(OAc) 2) b) centrifuged at 10 5,000 x g ribosomal p e l l e t S100A a) mixed with buffer C (1 mM Mg(OAc) 2) b) centrifuged at 105,000 x g ribosomal p e l l e t supernatant a) mixed with buffer D (5 mM Mg(OAc) 2) b) centrifuged at 105,000 x g ribosomal p e l l e t supernatant a) mixed with buffer E (10 mM Mg(OAc) 2) b) centrifuged at 105,000 x g c) repeated a) and b) 3x ribosomal p e l l e t [mixed with buffer supernatant F (10 mM Mg(OAc) 2) ribosomal suspension (WRib) a) Put on DEAE-cellulose column e q u i l i b r a t e d with buffer G and wash with same buffer b) eluted with buffer H ribosomal suspension centrifuged at 105,000 r ribosomal p e l l e t "I x g I mixed with buffer F ribosomal suspension (RSI) 1' supernatant * Buffer A-F described previously i n L i s t of Buffers. 48 An inoculum was prepared from the c o l i mutant grown on the surface of an agar s l a n t as i n (b) above. When growth has reached the mid-log phase, the inoculum was added to a 1 5 - l i t e r carboy containing the same medium as (b) above except that 4 yg u r a c i l / m l medium was added. Where l a b e l l e d c e l l s were required, 3 H - l a b e l l e d u r a c i l was added to the carboy p r i o r to log phase i n presence of the same amount of unlabelled u r a c i l . The c e l l s were grown and harvested i n the same manner as (b) above and f i n a l l y stored at -20°. Preparation of E. c o l i B Ribosomes (WRib) (see Chart I) Ribosomes were prepared from E_^  c o l i by a combination of many methods (218-220) which were intended to remove a l l non-ribosomal material. This was achieved by employing a seri e s of buffers with the same high ammonium concentration but with varying magnesium concentrations. A l l operations were performed at 0°-4° . Frozen or fresh c e l l s were ground with three times (3x) the weight of Superbrite glass beads (3M Company, previously cleaned i n 6NHC1 and washed with d i s t i l l e d water) by means of a V i r t i s 45 homogenizer running at top speed i n the presence of an equal volume of buffer A (10 mM T r i s , 10 mM Mg(OAc) 2» lOmM NHi,Cl, 10 mM mercaptoethanol, pH 7.8). The c e l l s were homogenized for four 2 minute i n t e r v a l s with f i v e minute cooling periods i n between. The homogenizing f l a s k was surrounded by ice throughout these operations. Following the l a s t homogenization, the homogenate was centrifuged at 10,000 x g for 20 mins i n order to remove 49 c e l l u l a r debris. The supernatant f r a c t i o n was c o l l e c t e d and 3 yg of pancreatic DNase I was added with gentle mixing to each m i l l i l i t e r of c o l i extract and incubated for 10 mins. The extract was then centrifuged at 30,000 x g for 20 mins. The supernatant s o l u t i o n was removed and the c e n t r i f u g a t i o n repeated. The upper f o u r - f i f t h s of the supernatant s o l u t i o n was removed and centrifuged at 105,000 x g for 5 hrs i n a Model-L u l t r a c e n t r i f u g e . The supernatant was removed and dialyzed versus buffer A overnight (30:1 ratio) and then stored at -70° . This was l a b e l l e d S-100 and i s the source of aminoacyl-tRNA synthetases and factors required for protein synthesis and was used i n many subsequent analyses. The ribosomal p e l l e t was dissolved i n buffer B (10 mM T r i s , 0.1 mM Mg(OAc) 2f 0.5-1.0 M NHi,Cl, pH 7.4) and mixed f o r 2 hrs. Aggregates were removed by centrifugation at 10,000 x g for 5 mins and then the ribosomal mixture was centrifuged at 105,000 x g for 15-24 hrs. The supernatant, S1Q0A, was saved and stored at -70°. This i s the source of i n i t i a t i o n and transfer factors required for protein synthesis. The ribosomal p e l l e t was dissolved i n buffer C (10 mM T r i s , 1 mM Mg(OAC) 2, 0.5-1.0 M NHWC1, pH 7.4) and mixed for 2 hrs. Aggregates were removed by ce n t r i f u g a t i o n at 10,000 x g for 5 mins and then the ribosomal mixture was centrifuged at 105,000 x g for 10-15 hrs. The supernatant was discarded and the ribosomal p e l l e t dissolved i n buffer D (10 mM T r i s , 5 mM Mg(OAc) 2, 0.5-1.0 M NtUCl, pH 7.4) and mixed for 2 hrs. Aggregates were removed by ce n t r i f u g a t i o n at 10,000 x g for 50 5 mins. and then the ribosomal mixture was centrifuged at 105,000 x g for 7-10 hours. The supernatant was discarded and the ribosomal p e l l e t dissolved i n buffer E (10 mM T r i s , 10 mM Mg(OAc) 2, 0.5-1.0 M NH„C1, pH 7.4) and mixed for 2 hrs. Aggregates were removed by ce n t r i f u g a t i o n at 10,000 x g for 5 mins and then the ribosomal mixture was centrifuged at 105,000 x g for 5 hrs. The supernatant was discarded and the mixing of the p e l l e t with buffer E and subsequent centrifuga-t i o n was repeated three times more. The ribosomal p e l l e t was f i n a l l y dissolved i n buffer F (10 mM T r i s , 10 mM NtUCl, 10 mM Mg(OAc) 2, pH 7.6) and mixed for 2 hrs. Aggregates were removed by c e n t r i f u g a t i o n at 10,000 x g for 5 mins. The ribosomal mixture (WRib) was checked for amino acid acceptor a c t i v i t y (the method of which w i l l be outlined i n d e t a i l below) and f i n a l l y stored at -70° i n small 4-ml v i a l s containg 1 ml a l i q u o t s . Assay f o r Ribosomal A c t i v i t y and Amino Acid Incorporation Ribosomal a c t i v i t y was determined by following the uptake of 1 "*C-Phe i n a poly U-dependent phenylalanine incorporation system. The assay system contained the following components ( t o t a l volume 250 yl) : 50 mM T r i s , pH 7.6 14 mM Mg(OAc) 2 100 mM NH„C1 6 mM ATP 2 mM GTP 100 nM unlabelled Phe 5 y l ^C-Phe (1:2 with H20) 50 yg poly U 50 y l WRib 100 y l S100 10 y l S100A 51 The previously frozen components were thawed and then placed on i c e and a l l additions were made at 0°-4°. The S100 and S100A v i a l s were discarded a f t e r each usage. A f t e r a l l additions were completed (the l l ,C-Phe was added l a s t 1 ) the tubes were incubated at 37° f o r 15 mins. After the incubation the tubes were quickly placed i n i c e and mixed with 3 ml of i c e cold TCA ( t r i c h l o r o a c e t i c a c i d ) . The tubes were then heated at >90° f o r 20 mins. They were then removed and kept at 0° for 15 mins. The protein 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 dried under a heating lamp. The dried f i l t e r s were placed i n s c i n t i l l a t i o n v i a l s and 5 ml of s c i n t i l l a t i o n f l u i d [containing 3g of PPO(2,5-diphenyloxazole) and 0.1 g of dimethyl-POPOP(l,4-bis-2-(4-methyl-5-phenyloxazoly1)-benzene per l i t e r of toluene] was added and counted i n a s c i n t i l l a t i o n counter. Assay for Amino Acid Acceptor A c t i v i t y The assay system was e s s e n t i a l l y that of K e l l e r (229) and was as follows : 0.2 ml of assay mix (250 mM T r i s , 100 mM Mg + +, 12.5 mM ATP, pH 7.6) 0.7 ml tRNA 0.1 ml enzyme (freshly prepared) 5 u l ^ C-amino acid mixture (1:2 with H 20) A l l a d d i t i o n s 2 were made at 0°-4°. The tubes were incubated at 37° f o r 20 mins. A f t e r the incubation the tubes were quickly placed i n i c e and mixed with 2 mis of i c e cold TCA to stop the reaction. The contents 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 placed i n s c i n t i l l a t i o n v i a l s and dried with the aid of a heat lamp. Five mis. of toluene 1 ' 2 See Appendix, page 163 52 s c i n t i l l a t i o n f l u i d was added and the v i a l s were then counted on a s c i n t i l l a t i o n counter. Preparation of E. c o l i Aminoacyl-tRNA synthetases E. c o l i aminoacyl-tRNA synthetases were prepared by a two-step procedure. Step (a) was employed to remove a l l the tRNA while step (b) was used to remove a l l low molecular weight material. In t h i s way a r e l a t i v e l y clean preparation of synthetases could be i s o l a t e d , (a) Treatment on DEAE-cellulose column A fiberous form of the r e s i n , DE-22 was used. The DE-22 was washed with IN NaOH and then with IN HC1 by the standard procedures as described i n the Whatman information l e a f l e t . DE-22, i n the f u l l y protonated form, was eq u i l i b r a t e d at the pH meter by s t i r r i n g with enough NaOH so that the pH on continued s t i r r i n g remained at about 7. This produces a material with a high capacity to bind tRNA (mean small ion capacity 1.0 meq/g dry r e s i n ) . Each mg of tRNA requires a column volume of 0.15 ml. The column was used only once and then the r e s i n was discarded. The following steps were a l l c a r r i e d out at 0°-4°. Three mis of S100 (approximately 0.6 mg RNA/ml) was thawed out and 0.75 ml of 1.0 M KC1 was added to give 3.75 ml of 0.2 M KCl. This enzyme mixture was then put through a 0.5 ml DE-22 column previously e q u i l i b r a t e d with 10 ml buffer G (0.2 M KCl, 10 mM T r i s , pH 7.4) . The f i r s t 0.4 ml of e f f l u e n t (the void volume) was discarded and then the next 3.7 mis was co 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 described i n the Pharmacia handbook, "Sephadex-gel f i l t r a t i o n i n theory and pract i c e . " The following steps were c a r r i e d out at 0°-4°. A 20 ml Sephadex G-25 column was prepared and e q u i l i b r a t e d with 35 ml of fr e s h l y prepared buffer H (20 mM T r i s , 10 mM cysteine, pH 7.4). Three mis of e f f l u e n t from (a) above was eluted through the column with buffer H. C o l l e c t i o n of e f f l u e n t was started as soon as the e f f l u e n t from (a) was pipetted onto the top of the column. Protein started to emerge from the column at the void volume which was about 35% of the column volume. Since the void volume varied with the packing of the column, small aliquots were removed from the column and tested with IM HClOi,. A d i s t i n c t t u r b i d i t y occurred when the concentration of protein i n the t e s t s o l u t i o n was 0.2 mg/ml or more. At th i s point a further 4.2 ml of 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% with respect to gl y c e r o l and stored at -20°. I t has been found that c e r t a i n aminoacyl-tRNA synthetases maintained t h e i r a c t i v i t y when stored for one year i n g l y c e r o l . In the case of seryl-tRNA synthetase the a c t i v i t y increased i n the presence of g l y c e r o l while with some others the a c t i v i t y decreased i n absence of gl y c e r o l (279) . The enzyme mixture was used as prepared without p r i o r removal of the g l y c e r o l . The column was washed 54 with 30 ml of d i s t i l l e d water to wash out the buffer and was ready for use again. Preparation of Benzoylated DEAE-cellulose (BD-cellulose) BD-cellulose ( f u l l y benzoylated), prepared by reaction of DEAE-cellulose with benzoyl chloride as described by Gillam et a l . (228) was ground and sieved i n the wet state through a 50 mesh (0.3 mm opening) screen and freed of f i n e p a r t i c l e s by repeated s e t t l i n g and decantation. During the sieving and the removal of the f i n e s , the BD-cellulose was maintained i n solutions containing d i l u t e (0.1-0.5 M) NaCl to prevent excessive generation of f i n e s . The BD-cellulose was packed int o columns by adding a s l u r r y of the r e s i n i n 2 M NaCl (which had been freed of trapped a i r by evacuation) to a column h a l f f i l l e d with 2 M NaCl. The s l u r r y was allowed to s e t t l e u n t i l approximately 2-3 cm of BD-cellulose had packed. The column stop-cock was then opened but the l i q u i d l e v e l was always kept above the packed surface of the exchanger. The s l u r r y was added u n t i l the desired depth of bed was obtained. The packed column was washed with 2 M NaCl u n t i l the eluate had acceptably low absorbance (A 26o nm 0.025). Following these washing steps, the column was e q u i l i b r a t e d with the s o l u t i o n used to s t a r t the e l u t i o n and the tRNA (dissolved i n the l a t t e r s o l u t i o n or a solution of equal or les s conductivity) was applied to the column. After a b r i e f r i n s i n g with 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 gradients of NaCl were applied i n the usual 55 manner (292, 293) . Treatment of Ribosomes with Puromycin The following incubation mixtures (in a t o t a l volume of 5 ml) were prepared and made 0.5 mM with respect to puromycin (PM) : A. 10 mM T r i s 6 mM ATP 14 mM Mg(OAc) 2 100 mM NIUC1 2 mM GTP 2 y l S100 100 y l S100A 46 mg WRib 200 mM amino acid B. 10 mM T r i s 6 mM ATP 14 mM Mg(OAc) 2 100 mM NH^Cl 2 mM GTP 100 y l S100A 20 mg WRib 2 ml S100 mix C. 5 mM T r i s 14 mM Mg(OAc) 2 100 mM NHUC1 2 mM GTP 100 y l S100A 13 mg WRib Incubation mixture A (148, 272) was brought up to pH 7.6 and preincubated for 5 mins at 37° p r i o r to PM addition and then further incubated for 30 mins at 37°. Following the incubation the mixture was centrifuged at 105,000 x g for 5 hrs. The p e l l e t was then mixed i n buffer I containing 10 mM T r i s , 0.1 mM Mg(OAc) 2, pH 7.6 and dialyzed overnight against the same buffer. A f t e r c e n t r i f u g a t i o n at 105,000 x g, the p e l l e t was mixed i n 10 mM Mg(OAc) 2 buffered at pH 7.8 (buffer A)and dialyzed overnight against the same buf f e r . Incubation mixture B (148, 272) was brought up to pH 7.6 and then incubated at 30° for 15 mins while incubation mixture C (148, 272) was brought up to pH 7.6 and incubated at 30° fo r 15 mins. Following the incubation both mixtures were made one molar with respect to NHi,Cl and centrifuged at 105,000 x g for 5 hrs. The p e l l e t s were mixed i n 10 mM Mg(OAc) 2 56 buffered at pH 7.6 (buffer F) and stored at -20°. Treatment of Ribosomes with Periodate ( H I O i » ) The WRib were incubated for two hours at 37° i n a mild a l k a l i n e b u f f e r (0.5 M T r i s . pH 9.0). This process stripped the bound tRNA of amino acids (273, 294). The l i b e r a t e d amino acids were removed by d i a l y s i s against d i s t i l l e d water at 4°. The ribosomal p e l l e t was obtained by cen t r i f u g a t i o n at 105,000 x g and mixed i n a buffe r containg 100 mM KOAc, pH 5.0 plus 1.1 umoles NalOif/mg RNA. This mixture was incubated at room temperature for 45 minutes i n the dark. A f t e r the incubation period 1.0 ml of g l y c e r o l was added to reduce any unreacted periodate and the mixture was reincubated fo r an ad d i t i o n a l 30 mins. The mixture was spun at 105,000 x g and the p e l l e t mixed i n 10 mM Mg(OAc)2 buffered at pH 7.6 (buffer F) . A n a l y t i c a l Polyacrylamide Gel Electrophoresis (a) A modification of the Davis system was used (221). The following stock solutions were prepared and stored i n brown bo t t l e s i n the cold room. Stock Solutions (A) IN HC1 48 ml (B) IN HC1 48 ml T r i s 36.6 gm TEMED 0.23 gm 8M urea to 100 ml T r i s 5.98 gm TEMED 0.46 gm 8M urea to 100 ml (pH 8.9) (pH 6.7) 57 Stock Solutions continued (C) acrylamide 28.0 gm Bis 0.735 gm 8M urea to 100 ml (E) r i b o f l a v i n 4 mg 8M urea to 100 ml (D) acrylamide 10.0 gm Bis 2.5 gm 8M urea to 100 ml (F) sucrose 40 gm 8M urea to 100 ml The following working solutions were prepared the day of the run and then discarded. Small-pore so l u t i o n #1 Small-pore so l u t i o n #2 Large-pore solut i o n Stock buffer s o l u t i o n fo r r e s e r v o i r s * 1 part A ammonium persulfate 2 parts B T r i s 6.0 gm 2 parts C 0.14 gm 4 parts D glycine 28.8 gm 1 part 7M urea 7M urea to 100 ml 2 parts E water to 1 l i t e r pH 8.9 2 parts F pH 8.3 2 parts sample * d i l u t e d 1:10 with water The gels were prepared i n c y l i n d r i c a l glass tubes, 0.5 x 10 cms. The clean glass tubes were f i r s t placed i n an upright p o s i t i o n i n a tube stand. Stands are conveniently made by cementing hollow rubber stoppers, for example, the B-D Vacu-tainer stoppers, i n a single row, a few cms apart, with the closed end down, to a f l a t piece of p l a s t i c . The open end of the cap should f i t snugly around the gel tube to prevent leakage of the ingredients. The large pore s o l u t i o n was prepared and run into the glass tubes. A water layer was then placed on top of the gel s o l u t i o n i n such a manner as not to disturb the gel surface. The tube stand was now placed d i r e c t l y under a fluorescent bulb for about 30 mins to allow f o r photopoly-58 merization. Following photopolymerization the water layer was removed and the gel tubes completely f i l l e d with small-pore gel s o l u t i o n , prepared by mixing equal volumes of small-pore solutions #1 and #2. The gels were allowed to polymerize while being protected from strong l i g h t . Following polymerization of the gels, the glass tubes were removed from the tube stand and placed i n the cold room on an electrophoretic apparatus, as i l l u s t r a t e d by Davis (221), containing the appropriate b u f f e r . The electrodes were attached to the apparatus and to a power supply set at a constant current of 5 mA/tube. P r i o r to turning on the power supply, a drop or two of 0.001% bromophenol blue was added to serve as the marker dye. A f t e r the run the glass tubes were removed from the el e c t r o p h o r e t i c apparatus and the gels were subsequently removed by rimming the tubes under water with a wire. The gels were stained for 1 hr i n a 15% IlAc s o l u t i o n containing 2% acridine orange ( s p e c i f i c for nucleic acids) and 1% lanthanum acetate ( f i x a t i v e ) (222). The gels were destained overnight i n 15% HAc and then stored i n 7% HAc i n the cold room. (b) A modification of the Dingman and Peacock system was used (223-225). The following working s o l u t i o n for the preparation of a 10% polyacrylamide gel was prepared the day of the run and then discarded: 59 7.9 ml acrylamide mixture (19.5% acrylamide + 0.5% Bis) 4.65 ml H 20 0.95 ml DMAPN (6.4%) 1.5 ml buffer (undiluted) (Tris 108 gm, EDTA diNa 9.3 gm, Boric Acid 55 gm : to 1 l i t e r with H 20, pH 8.3) 10 mg ammonium persulfate The gels were prepared i n c y l i n d r i c a l glass tubes, 0.5 x 10 cms. The glass tubes were f i r s t placed i n an upright p o s i t i o n i n a tube stand. The gel mixture was prepared, mixed and run i n t o the glass tubes f o r a distance of 7 cms. A water layer was then placed on top of the gel s o l u t i o n i n such a manner as not to disturb the gel surface and the gels were allowed to polymerize. A f t e r polymerization, the water layer was removed, and the glass tubes were f i l l e d with d i l u t e d b u f f e r (1:10 with H 20). The tubes were then removed from the tube stand and placed i n the cold room on an elec t r o p h o r e t i c apparatus, as described i n (a), containing the same d i l u t e d b u f f e r . The tubes were then p r e e l e c t r o -phoresed f o r 30 mins at 5mA/tube. A f t e r preelectrophoresis, the buffe r was removed from the glass tubes and up to 200 y l of sample i n 10% sucrose, i n the presence of a drop or two of bromophenol blue, was layered onto the gel surface. The r e s t of the glass tube was f i l l e d with d i l u t e d b u f f e r so as not to disturb the sample layer and then placed i n the cold room on the electr o p h o r e t i c apparatus. The electrodes were attached and electrophoresis was c a r r i e d out at 10 v o l t s per cm length of glass tube. A f t e r the run the gels were obtained as described i n (a) 60 and then stained for 1 hr i n 0.2% methylene blue dissolved i n an acetate b u f f e r (0.4 M NaOAc, 0.4 M HAc, pH 4.7). The gels were destained overnight i n d i s t i l l e d water and then stored i n d i s t i l l e d water i n the cold room. This was found to be the best method for the separation of low molecular weight RNA and was subsequently used f o r the i d e n t i f i c a t i o n of these RNAs i n the Results section. (c) A modification of the method of Moriyama et a l . (195) was used. The only difference between t h i s procedure and that used i n (b) was the type of gel preparation. Preparation of the gel The following working s o l u t i o n for the preparation of a 10% polyacrylamide gel was prepared the day of the run and then discarded. 0.5 ml of 10% ammonium persulfate and 10 y l TEMED was added to 25 ml of 10% gel s o l u t i o n (9.75% acrylamide + 0.25% Bis i n 40 mM T r i s , 20 mM NaOAc, 2 mM EDTA, pH 7.4, 25°) previously degassed f o r a few seconds. Preparative Polyacrylamide Gel Electrophoresis A Canalco preparative d i s c electrophoresis apparatus with a PD-2/320 upper column was used (194). The following stock solutions were prepared and stored i n brown bo t t l e s i n the cold room. (A) (C) IN HC1 48 ml T r i s 36.6 g TEMED 0.23 g acrylamide 28.0 g Bis 0.735 g 8M urea to 100 ml 8M urea to 100 ml (pH 8.9) The following working solutions were prepared the day of the run and then discarded. Small-pore s o l u t i o n #1 Small-pore so l u t i o n #2 E l u t i o n buffers Stock buffer solu-tions f o r electrode compartments* 1 part A 2 parts C 1 part 7M urea pH 8.9 ammonium persu l f a t e 0.14 g 7M urea to 100 ml (1) 1 part A 7 parts H 20 (2) 40 mM T r i s 20 mM NaOAc 2 mM EDTA, (1) T r i s 6.0 g (2)upper electrode glycine 28.8 g H 20 to 1 l i t e r , pH 8.3 pH 7.2 buffer : 40 mM T r i s 20 mM NaOAc 2 mM EDTA, pH 7.2 lower electrode buffe r : 80 mM T r i s 40 mM NaOAc 4 mM EDTA, pH 7.2 • d i l u t e d 1:10 with H 20 In order to prepare the g e l , the bottom end of the upper column was capped with a square of 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 with the bottom end r e s t i n g on a f l a t surface. The capped upper column was f i r s t f i l l e d to the mark with small-pore gel so l u t i o n , prepared by mixing equal volumes of small pore solutions #1 and #2 and then a water-layer was placed on top of the gel so l u t i o n i n such a manner as not to disturb the gel surface. The column was then placed between two fluorescent bulbs. Following polymerization the water layer was removed along with the Saran Wrap and the upper column was f i l l e d i n t o the assembly. The sample i n 10% sucrose was c a r e f u l l y layered 62 on top of the gel surface. The upper electrode buffer was then slowly added, so as not to disturb the gel surface, u n t i l the upper column r e s e r v o i r was f i l l e d . The lower electrode r e s e r v o i r was also f i l l e d with the appropriate buffer and then both electrodes were connected to a power supply set at a constant current of 7mA. The appropriate e l u t i o n buffer was pumped i n and out at a flow rate of 60 ml per hr, and c o l l e c t e d by means of a f r a c t i o n c o l l e c t o r . The system was kept at 4° by means of two separate cooling systems. A marker dye was not used i n order to avoid the possible e f f e c t s of i t s c o n t r i -bution to the A 2 6 o readings of the c o l l e c t e d e f f l u e n t . The run was terminated when the A 2 6 o readings of the e f f l u e n t had become n e g l i g i b l e . Preparation of 30S and 50S subunits from Ribosomes The preparation of ribosomal subunits was e s s e n t i a l l y that of Cannon et a l . (226). The ribosomal p e l l e t was mixed with buffer K (5 mM T r i s , pH 7.3 + 0.1 mM Mg(OAc) 2) and d i a l y z e d against the same buffe r for 28 hrs. The ribosomal mixture was then put on a 10-30% discontinuous sucrose gradient which contained equal aliquots of 10, 15, 20, 25 and 30% sucrose prepared i n the same buffer and centrifuged at 25,000 rpm for 12 hrs at 4° i n a Model L u l t r a c e n t r i f u g e using a SW-39 rot o r . 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 c o l l e c t e d through the use of the Beckman f r a c t i o n recovery system. In t h i s procedure each tube was placed i n a tube holder and a recovery cap was screwed on top of the holder to 63 maintain an a i r - f r e e system. The tube was pierced from the bottom by a needle, dense sucrose was pumped i n slowly through the needle so as not to disturb the gradient and fr a c t i o n s were c o l l e c t e d from a rubber tube leading from the recovery cap. Preparation of CsCl Gradient The method was e s s e n t i a l l y that of Meselson e t a l . (227) and Traub and Nomura (6 0). Each of the ribosomal subunits was centrifuged through a CsCl gradient. The ribosomal subunits were dissolved i n a buffer made up of 20 mM T r i s , 40 mM MgCl 2, pH 7.6. One ml of each subunit suspension was mixed with 4.3 ml of 61% (w/v) CsCl dissolved i n the same bu f f e r . The r e s u l t i n g mixture had an index of r e f r a c t i o n r\2* = 1.39 50. Approximately 5.0 ml of th i s mixture was placed i n a Lusteroid centrifuge tube i n a Spinco SW-39 rotor. To insure the s t a b i l i t y of the subunits, i t was found necessary to wash the Lusteroid tubes. This was done for 1 hr i n b o i l i n g 1 mM EDTA and then i n b o i l i n g water. Centrifugation was c a r r i e d out at 36,000 rpm for 36 hrs at 4°. A f t e r slow deceleration, each Lusteroid tube was withdrawn and placed i n a tube holder (Beckman). The tubes were pierced from the bottom with a needle and fr a c t i o n s were c o l l e c t e d by gra v i t y . I s o l a t i o n of Tot a l RNA from Ribosomes Ribosomes were mixed with buffer L (10 mM T r i s , pH 7.6, 10 mM Mg + + + 0.5% sodium dodecyl s u l f a t e (SDS)) and an equal 64 volume of water-saturated phenol for 20 mins at room temperature. The aqueous layer was c o l l e c t e d a f t e r c e n t r i -fugation at 5,000 x g for 10 mins. The phenol layer was mixed with h a l f a volume of buffer for 20 mins. The aqueous layer was recovered a f t e r c e n t r i f u g a t i o n and pooled with the i n i t i a l aqueous lay e r . I t was then mixed with an equal volume of phenol for 20 mins at room temperature. The aqueous layer was recovered 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 . Residual ether was removed by bubbling N 2 through the s o l u t i o n . Sodium acetate (1.5 M, pH 5.2) was added to give a 2% so l u t i o n and the RNA p r e c i p i t a t e d by addition of 3 volumes of cold ethanol. 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 dissolved i n ei t h e r 10 mM Mg(OAc) 2 buffered at pH 7.6 (buffer F) or water. Preparation of High and Low Molecular Weight RNA from Ribosomes The t o t a l RNA from ribosomes was prepared as described above. The RNA was dissolved i n 0.1 M T r i s , pH 7.5 and then t h i s mixture was made 2 M with respect to NaCl and kept at 4° for 2 days. The p r e c i p i t a t e containing the high molecular weight RNA was separated from the supernatant containing low molecular weight RNA by low speed c e n t r i f u g a t i o n . In order to ensure a better separation of high and low molecular weight material, the RNA p r e c i p i t a t e was dissolved i n the above buffer and r e p r e c i p i t a t e d when the buffer was made 2 M with respect to NaCl. The p r e c i p i t a t e , containing high molecular weight RNA, was dissolved i n 50 mM NaCl and stored 65 at -20°. The supernatant was made 10 mM with respect to Mg(OAc) 2 and then NaOAc (1.5 M, pH 5.2) was added to give a 2% so l u t i o n . The RNA was subsequently p r e c i p i t a t e d with 3 volumes of cold ETOH. The low molecular weight RNA was c o l l e c t e d by cen t r i f u g a t i o n and dissolved i n 50 ml NaCl. I t was subsequently further characterized on Sephadex G-100 and by preparative gel electrophoresis. 66 supernatant - i a) mixed with buffer M (5mM Mg(OAc) 2/ 250mM NH^Cl b) Put on DEAE-cellu-lose column e q u i l i b r a t e with buffer M and wash with same buffer c) elute with buffer N (lOmM Mg(OAc) 2, 1.0M NH^Cl ribosomal p e l l e t _ a) mixed w i t i buffer G b) centrifuged at 105,000 x g a) mix with buffer H b) centrifuged at 105,000 x g c) steps a) and b) repeated 2x supernatant ribosomal p e l l e t (RSII) ribosomal suspension |centrifuged at 105,000 x g P - i ^ ribosomal p e l l e t supernatant mixed with ] buffer F ribosomal suspension (RSIII) I I i 66 CHART I I * Procedures f o r preparing E. c o l i ribosomes E. c o l i a) ground with glass beads i n buffer A (10 mM Mg(OAc) 2) b) centrifuged at 10,000 x g c e l l u l a r debris supernatant a) incubated with DNase b) centrifuged 2x at 30 ,000 x g p r e c i p i t a t e 1 supernatant centrifuged at 105 ,000 x g •ribosomal p e l l e t supernatant a) mixed with (S100) buffer B (0.1 mM Mg(OAc) 2) b) centrifuged at 105,000 x g ribosomal p e l l e t S100A a) mixed with buf f e r C (1 mM Mg(OAc) 2) b) centrifuged at 105,000 x g ribosomal p e l l e t supernatant a) mixed with buf f e r D (5 mM Mg(OAc) 2) b) centrifuged at 105,000 x g i ribosomal p e l l e t supernatant a) mixed with buffer E (10 mM Mg(OAc) 2) b) centrifuged at 105,000 x g c) repeated a) and b) 3x 1 ribosomal p e l l e t supernatant lmixed with buffer F (10 mM Mg(OAc) 2) I1 ribosomal suspension (WRib) a) Put on DEAE-cellulose column equ i l i b r a t e d with buffer M and wash with same buffer b) eluted with buffer N ribosomal suspension |centrifuged at 105,000 x g p i j ribosomal p e l l e t supernatant mixed with buffer F I1 ribosomal suspension (RSI) * Buffer A-H, M-N described previously i n L i s t of Buffers. 67 TABLE I The a c t i v i t y of ribosomes prepared by d i f f e r e n t methods Concentration Ribosome preparation* A 2 6 0 units A c t i v i t y * * Experiment I WRib 50 29 RSI 52 74 WRib 20 16 RSI 18 33 Experiment II 3H-WRib*** 11.6 23 RSII 10.9 20 RSIII 9.6 24 WRib 10.0 2 * The de s c r i p t i o n of the preparations are given i n Chart I I . ** A c t i v i t y determined by following the incorporation of 1 **C-Phe i n a poly U-directed synthesis of polyphenylalanine described i n Materials and Methods. The a c t i v i t y of the blank containing no ribosomes was set at one (200 cpm). *** This preparation i s i d e n t i c a l to WRib except the c e l l s originated from a pyrimidine-requiring mutant of E_^  c o l i B (ATCC 13135) grown i n the presence of 3 H - u r a c i l . 68 RESULTS The A c t i v i t y of Ribosome Preparations The ribosomes prepared as outlined i n Materials and Methods were found to have an amino ac i d acceptor a c t i v i t y 50-100 f o l d over the blank l e v e l which was set at one. The A 2 6 0 / A 2 8 0 r a t i o of these ribosomes was found to be 2:1 and was comparable and i n many cases greater to other reported preparations (282) . These ribosomes (WRib) were subsequently used i n a l l future experiments. Ribosomes were prepared by three other procedures as outlined i n Chart II and compared with WRib with respect to a c t i v i t y (Table I) and bound low molecular weight RNA (Figure 1). The RSI (Table I) which had been put through a DE-22 column was much more active than WRib. This a c t i v i t y was s t i l l less than that of the o r i g i n a l fresh preparation prepared two months e a r l i e r which showed 82-fold amino ac i d acceptor a c t i v i t y over the blank l e v e l . In experiment I, Table I, the a c t i v i t y of the ribosomes increased as the concentration of the ribosomes (measured i n A 2 6 o units) increased. In experiment I I , Table I, freshly prepared l a b e l l e d WRib from a pyrimidine-requiring c o l i mutant had only a 23-f o l d increase i n a c t i v i t y over the blank. Perhaps t h i s was due to the type of c e l l used, the stage of growth at which the c e l l s were harvested or the manner i n which the ribosomes were prepared (see Materials and Methods). At the p a r t i c u l a r concentration used, the 3H-WRib, RSII and RSIII had v i r t u a l l y FIGURE 1 Photograph of polyacrylamide gel (10% g e l , pH 8.3, and 0.5 x 7 cm) electrophoresis patterns of low molecular weight RNA obtained from d i f f e r e n t preparations of E^ c o l i ribosomes. From l e f t to r i g h t *: WRib, RSI, RSII, RSIII and the control (commercial E^ c o l i tRNA). * The abbreviations are those designated i n Chart I I . The 4S and 5S RNA regions indicated have been predetermined by numerous investigators and v e r i f i e d by the use of appro-p r i a t e standards (223-225). To face page 69 69 70 the same a c t i v i t y . The WRib which showed only s l i g h t a c t i v i t y was the same as t h a t used i n the f i r s t experiment, Table I , except t h a t i t had been thawed and f r o z e n one more time. Perhaps t h i s was one of the reasons f o r i t s d r a s t i c a l l y lowered a c t i v i t y . Figure 1 shows the polyacrylamide 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 low molecular weight RNA obtained from the 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 Chart I I . There appears t o be much l e s s 4S and 4.5S RNA* bound to ribosomes which have been washed through a DEAE-cellulose column w i t h 1 M NHi»Cl. This may 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 ribosomal a c t i v i t y observed under these c o n d i t i o n s (Table I ) . The r e g i o n designated X may be 6S RNA. Attempts to Remove Bound tRNA from Ribosomes The WRib were t r e a t e d under va r i o u s c o n d i t i o n s i n an e f f o r t t o remove a l l the 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. A f t e r the i n c u b a t i o n p e r i o d , the PM was removed w i t h 1 M NH^Cl. The c a p a c i t y of these PM-WRib to support p r o t e i n s y n t h e s i s was found to be at l e a s t as high as the untreated ones and i n some cases the a c t i -v i t y was found t o be c o n s i d e r a b l y h i g h e r . Figure 2 shows the * 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 i d e n t i -f 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 of polyacrylamide gel (10%, pH 8.3, and 0.5 x 7 cms) electrophoresis patterns of low molecular weight RNA obtained from puromycin-treated c o l i ribosomes (PM-WRib). From l e f t to r i g h t : PM-WRib (preparation B i n Materials and Methods), PM-WRib (preparation C), PM-Mg treated WRib (preparation A), PM-Mg treated WRib (preparation A but with twice the PM concentration) and the control (commercial E. c o l i tRNA). To face page 71 71 72 separation of low molecular weight RNA from the various PM-WRib on polyacrylamide gels. These PM-WRib were found to contain bound tRNA. I n some experiments a f t e r the incubation period with PM, these PM-WRib were subsequently suspended i n 0.1 mM Mg(OAc) 2 buffered at pH 7.6 (buffer I) and dialyzed against the same bu f f e r (referred to as PM-Mg treated WRib). These PM-Mg treated WRib were found to have very low a b i l i t y to support protein synthesis and the resu l t s i n Figure 2 showed that there was only a trace amount of tRNA bound to these PM-Mg treated WRib. This experiment was repeated using a higher PM concentration, 1 mM and then these PM-WRib were subsequently suspended and dialyzed against b u f f e r I . A n e g l i g i b l e amount of bound tRNA was detected i n these PM-Mg treated WRib (Figure 2). These ribosomes were also found to have very low amino acid acceptor a c t i v i t y . * (b) Periodate WRib were treated with HIOi» i n 100 mM KOAc, pH 5.0 a f t e r p r i o r mild a l k a l i n e hydrolysis to s t r i p amino acids from bound tRNA as described i n Materials and Methods. Unfortun-ately HICU completely disrupted the ribosomal structure such that the c h a r a c t e r i s t i c ribosomal p e l l e t could not be obtained even a f t e r extended periods of u l t r a c e n t r i f u g a t i o n . There-fore t h i s procedure had to 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 of polyacrylamide gel (10%, pH 8.3 and 0.5 x 7 cm) electrophoresis patterns of low molecular weight RNA obtained from Mg-treated E^ c o l i ribosomes. From l e f t to r i g h t : Mg-treated WRib (0.1 mM Mg + +), Mg-treated WRib (0.1 mM Mg + + followed by 10 mM Mg + +) and the control (commercial E^ c o l i tRNA) . To face page 73 73 FIGURE 4 The sedimentation pattern of c o l i ribosomes on a 10-30% discontinuous sucrose gradient. The ribosomes (200A 26o units) were f i r s t suspended i n buffer K, then dialyzed against the same buffe r and subsequently centrifuged through a sucrose gradient at 25,000 rpm for 12 hrs at 4°. One ml f r a c t i o n s were c o l l e c t e d . A complete d e s c r i p t i o n i s given i n Materials and Methods. To face page 74 74 FRACTION NUMBER 75 (c) D i a l y s i s against 0.1 mM Magnesium WRib were mixed i n a buffer containing 5 mM T r i s , pH 7.4 plus 0.1 mM Mg(OAc) 2 and dialyzed against the same buffer for 2 days with frequent changes i n the buf f e r . The ribosomal p e l l e t was c o l l e c t e d by cen t r i f u g a t i o n , mixed with 10 mM Mg(OAc) 2 buffered at pH 7.6 (buffer F) and an al i q u o t was stored at -20°. The re s t of the suspension was dialyzed against the same buffer overnight and then subsequently frozen at -20°. Electrophoresis i n a 10% polyacrylamide gel of the low mole-cular weight RNA from these Mg + +-treated WRib i s shown i n Figure 3. The WRib dialyzed only against low Mg + + showed a strong 5S RNA band but very f a i n t 4.5S and 4S RNA bands r e l a t i v e to the 5S RNA. The WRib dialyzed against low and then high Mg + + showed a dense 5S RNA band and a strong 4.5S RNA band but n e g l i g i b l e 4S RNA band r e l a t i v e to the 5S RNA. I t appears that d i a l y s i s against low Mg"*""1' removes v i r t u a l l y a l l the tRNA normally bound to WRib. Studies on the 30S And 50S Ribosomal Subunits E. c o l i ribosomal subunits were prepared as described i n Materials and Methods. Figure 4 shows the separation of the subunits on a 10-30% discontinuous sucrose gradient. Fractions were c o l l e c t e d from the top as described i n the text. The 4S RNA was i d e n t i f i e d as tRNA by the f a c t that i t had amino acid acceptor a c t i v i t y . Figure 5 shows the re s u l t s of e l e c t r o -phoresis of the RNA from the subunits i n a 10% polyacrylamide g e l . The gel containing only the RNA from the 30S ribosomal FIGURE 5 Photograph of polyacrylamide gel (10%, pH 8.3 and 0.5 x 7 cm) electrophoresis patterns of low molecular weight RNA obtained from E_;_ c o l i ribosomal subunits. From l e f t to r i g h t : 30S RNA*, CsCl-treated 30S RNA**, 50S RNA*, 50S RNA (5% g e l ) * , 50S RNA*, CsCl-treated 50S RNA**, tRNA from sucrose gradient and the control (commercial E^ c o l i tRNA). * Represents the t o t a l RNA from the 30S and 50S ribosomal subunits r e s p e c t i v e l y . ** The 30S ribosomal subunit was centrifuged through a CsCl gradient as described i n Materials and Methods and then the t o t a l RNA was i s o l a t e d from these subunits as previously described. The 50S ribosomal subunit was treated s i m i l a r l y . To face page 76 7 6 FIGURE 6 Sedimentation pattern of E^ c o l i ribosomal subunits i n a CsCl s o l u t i o n . The subunits were prepared as described i n Figure 4 and then centrifuged at 36,000 rpm for 36 hrs at 4° i n 61% (w/v) CsCl as described i n Materials and Methods. A. Represents the sedimentation of 30S ribosomal subunits (69A 26o units) through CsCl. One ml fr a c t i o n s were c o l l e c t e d and 5 y l aliquots were removed for the absorbance readings. B. Represents the sedimentation of 50S ribosomal subunits (483A 2 6o units) through CsCl. One ml f r a c t i o n s were c o l l e c t e d and 5 y l aliquots were removed for the absorbance readings. To face page 77 FIGURE 7 E l u t i o n pattern of 100 mg of commercial E^ c o l i tRNA from Sephadex G-100. The RNA was chromatographed on a 3 x 200 cm column and eluted with 50 mM NaCl at a flow rate of 6 ml/hr. Five ml fract i o n s were c o l l e c t e d and one ml aliquots were removed f o r absorbance readings. To face page 78 40 60 80 100 120 FRACTION NUMBER 140 160 180 200 79 subunit shows only one dense region at the top of the gel in d i c a t i n g that i t contains only high molecular weight RNA. The gel containing only the RNA from the 50S ribosomal subunit also has a dense region at the top of the gel but i n addition a double 5S RNA band with what appears to be a trace of 4.5S and 4S RNA. The gel containing the tRNA f r a c t i o n from the sucrose gradient also shows a d i s t i n c t band i n the 5S region. The subunits were each put through a CsCl gradient as described i n Materials and Methods. Figure 6 shows the sedimentation density pattern of each of the CsCl-treated subunits. Each shows a single peak. The CsCl-treated subunits were then electrophoresed i n a 10% polyacrylamide g e l . The r e s u l t s are shown i n Figure 5. The gel containing only the RNA from CsCl-treated 30S ribosomal subunits shows a number of bands from the top of the gel to the 5S RNA region where there appears to be a trace band. The corres-ponding gel containing the RNA from CsCl-treated 50S r i b o -somal subunits shows a strong 5S RNA band and d e f i n i t e 4.5S and 4S RNA bands. Characterization of the Ribosomes Figure 7 shows the o p t i c a l density pattern of commercial E. c o l i tRNA (General Biochemicals) fractionated on a long Sephadex G-100 column (200 x 3 cm). The f i r s t peak i s high molecular weight RNA, the second peak 5S RNA and the t h i r d peak, the major peak, contained the 4S RNA material. Figure 8 shows a 10% polyacrylamide gel pattern of peaks II and I I I FIGURE 8 Photograph of polyacrylamide gel (10%, pH 8.3 and 0.5 x 7 cm) electrophoresis patterns 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 previously on a Sephadex G-100 column (see Figure 7). From l e f t to r i g h t : peak I I , peak III and the control (commercial E. c o l i tRNA). To face page 80 80 FIGURE 9 E l u t i o n pattern of t o t a l ribosomal RNA (8300A 26o units) from E. c o l i on Sephadex G-100. The RNA was fractionated on a 3 x 200 cm column and eluted with 50 mM NaCl at a flow rate of 6 ml/hr. Tubes 1-25 contain 20 ml and a l l subsequent tubes contain 5 ml f r a c t i o n s . One ml aliquots were removed for absorbance readings. S o l i d l i n e : A 2 6 o; dotted l i n e : 1 "*C-amino acid acceptor a c t i v i t y . To face page 81 FRACTION NUMBER 82 TABLE II The D i s t r i b u t i o n of RNA i n E. c o l i Ribosomes Peak F r a c t i o n Number* T o t a l A 2 6 o Units % of Total I 2 0 - 4 3 7897 97.2 II 45 - 64 128 1.5 III 65 - 120 103 1.3 The f r a c t i o n s are those shown i n Figure 9. 83 from Figure 7. The gels were run using an aliq u o t from the combined f r a c t i o n s within each peak region. The 4S RNA f r a c t i o n (peak III) i s well separated from the 5S RNA f r a c t i o n (peak II) although i t does contain a trace of 4.5S RNA. The 5S RNA f r a c t i o n also contains the major part of the 4.5S RNA and a trace of 4S RNA material. The reason trace amounts of other types of RNA were found i n both peaks was because peak tubes were not used i n the electrophoresis runs. This w i l l be c l e a r l y demonstrated further on i n the t h e s i s . Figure 9 shows the f r a c t i o n a t i o n of the t o t a l RNA obtained from c o l i WRib run on the same Sephadex G-100 column. The major peak i s high molecular weight ribosomal RNA, the second peak contains 5S rRNA and the t h i r d peak, 4S RNA as confirmed by acceptor studies. The l a s t peak con-tained r e s i d u a l phenol l e f t over from the procedure used to obtain the t o t a l ribosomal RNA which was described i n Materials and Methods. Table II shows the amount of RNA i n each peak region. The A 2 6 0 r a t i o of 4S : 5S RNA i s 0.87 which indicates that there are approximately 1.3 molecules of 4S RNA bound to the ribosomes per molecule of 5S RNA*. Figure 10 shows the separation of peaks II and III from Figure 9 by electrophoresis i n 10% polyacrylamide gels. The gels were run using 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 calcu-lated given the molecular weight of SS and 4S RNA as 4 x 103 and 2.5 x IO3 respectively. FIGURE 10 Photograph of polyacrylamide gel (10%, pH 8.3 and 0.5 x 7 cm) electrophoresis patterns of peaks II and III as designated i n Table I I . From l e f t to r i g h t : peak I I , peak I I I and the co n t r o l (commercial E. c o l i tRNA). To face page 84 84 85 TABLE I I I S p e c i f i c amino ac i d acceptor a c t i v i t y of tRNA bound to E^ c o l i ribosomes 3 Ribosomal Numbers of bound tRNA Tot a l tRNA* known s p e c i f i c amino acid p moles/A 2 6 o p moles/A2 6o tRNAs i n ] unit u n i t A l a 13.4 2 Arg 60.0 2 Asn 46 .4 (19.0) 3 Asp 43.4 2 Gly 16 .1 (15.2) 3 Glu 37.8 2 His 45.7 3 H e 14.8 2 Leu 109 .4 106.9 5 Lys 108.5 (59.0) 2 Met 209 .0 73.6 2 Phe 65.5 42.7 3 Pro 33.0 (68.9) 4 Ser 26.3 72.3 (43.4) 4 Thr 65.1 3 Trp 383.0 27.3 5 Tyr 12.7 43.6 (29 .3) 2 V a l 163.6 (76.4) 2 Results shown i n brachets were done i n t h i s laboratory by R. Chase. The other r e s u l t s i n th i s column are those of Bartz et a l . (291) . T o t a l RNA refers to cytoplasmic and ribosomal bound tRNA. References 274, 275. See Appendix, page 163. FIGURE 11 E l u t i o n pattern of the low molecular weight RNA (1368A 26o units) bound to E^ c o l i ribosomes from Sephadex G-100. The RNA was fractionated on a 3 x 200 cm column and eluted with 50 mM NaCl at a flow rate of 6 ml/hr. Five ml fracti o n s were c o l l e c t e d . S o l i d l i n e : A 2 6 o ; dotted l i n e : 1'*C-amino acid acceptor a c t i v i t y . To face page 86 86 FRACTION NUMBER 87 from the combined f r a c t i o n s shown i n Table I I . The r e s u l t s 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 Figure 8. The dotted l i n e s i n Figure 9 indicate the region of amino acid acceptor a c t i v i t y . The peak tubes were pooled and acceptor studies were done on a l l the amino acids except glutamine and cysteine. The r e s u l t s are shown i n Table I I I . E_^_ c o l i ribosomes have bound tRNA which has acceptor a c t i v i t y for a l l the amino acids. The amount of changing varies from one tRNA species to another. Another experiment was c a r r i e d out i n order to get more d e f i n i t i v e data on the d i s t r i b u t i o n of the major low molecular RNA species bound to ribosomes. The t o t a l ribosomal RNA was i s o l a t e d from WRib using the phenol technique. The recovery of rRNA by t h i s procedure was 66.3% on the basis of the t o t a l A 2 6 0 units recovered from the s t a r t i n g material. The rRNA was dissolved i n 100 mM T r i s , pH 7.5 and the s o l u t i o n was made 2 M with respect to NaCl and kept at 4° for a day. The supernatant, which contained the low molecular weight RNA, made up 9% of the t o t a l ribosomal RNA. This RNA was p r e c i p i -tated with cold ETOH, dissolved i n 0.05 M NaCl, loaded into a Sephadex G-100 column (3 x 200 cm) and eluted with the same buff e r . The r e s u l t s are shown i n Figure 11. The major peak contained high molecular weight RNA, the second peak contained the 5S RNA and the minor peak, the 4S RNA as determined by acceptor studies. The dotted l i n e s indicate the region of amino acid acceptor a c t i v i t y . The recovery of RNA from the 88 TABLE IV D i s t r i b u t i o n of low molecular weight RNA i n E. c o l i ribosomes i s o l a t e d by Sephadex G-100 chromatography* T o t a l Actual % bound F r a c t i o n A 2 6 o units % of To t a l to ribosomes Peak I 656 50.7 95.5 Peak II 404 31.2 2.8 Peak III 230 18.1 1.7 * The data are from Figure 11. FIGURE 12 Photograph of polyacrylamide gel (10%, pH 8.3 and 0.5 x 7 cm) electrophoresis patterns of the peak f r a c t i o n of each peak region of Figure 11. From l e f t to r i g h t : f r a c t i o n 86, f r a c t i o n 116, f r a c t i o n 132, fra c t i o n s 116 and 132 and the control (commerical E. c o l i tRNA). b To f a c e page 89 89 FIGURE 12a Chromoscan tracing of a mixture of fr a c t i o n s 116 and 132 (peaks II and III) from Figure 11. Equal concentrations of both f r a c t i o n s were mixed. To face page 90 91 column was 95%. Table IV shows the d i s t r i b u t i o n of RNA i n these peak regi o n s . The A 2 6 o r a t i o of 4S : 5S RNA was approximately 0.58 : 1 which i n d i c a t e s t h a t there i s a p p r o x i -mately 0.90 molecule of 4S bound to the ribosomes per molecule of 5S RNA*. The peak f r a c t i o n i n each peak r e g i o n of F i g u r e 11 was run on a 10% polyacrylamide g e l . The r e s u l t s are shown i n F i g u r e 12. Peak I ( F r a c t i o n 86) contained one major band which remained a t the top of the g e l and had presumably a molecular weight i n excess of 6S. Peak II ( f r a c t i o n 116) contained 3 major bands - a double 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) contained the bands corresponding to 4S RNA. 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 116 and 132 (peaks II and III) were combined and e l e c t r o -phoresed i n a 10% polyacrylamide g e l (Figure 12). This g e l was subsequently scanned i n a Joyce-Loebl Chromoscan. The r e s u l t s are shown i n F i g u r e 12a. The 4S RNA appears as a probably double band. The m a t e r i a l preceding 4S RNA is^degraded RNA. Without t a k i n g the degraded RNA i n t o account, the 4.5S RNA represents 2.7% of the t o t a l RNA. Even though t h i s 4.5S RNA i s spread throughout the 4 to 5S RNA r e g i o n , i t s t i l l r e p r e -sents much l e s s than one molecule per molecule of 5S RNA. Other Techniques used to F r a c t i o n a t e Low M o l e c u l a r Weight RNA (a) P r e p a r a t i v e G el 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 ribo-some 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 103 and 2.5 x 103 respectively. FIGURE 13 Electrophoretic pattern i n a 10% preparative polyacrylamide gel of 5 mg of commercial E_^ c o l i tRNA. The f r a c t i o n a t i o n procedure i s given i n Materials and Methods. Flow rate was 60 ml/hr and 5 ml fra c t i o n s were c o l l e c t e d . One ml fra c t i o n s were removed for absorbance readings. To face page 9 2 92 I L_ I I I I l I 0 10 20 30 40 50 60 FRACTION NUMBER FIGURE 14 Electrophoretic pattern of 100 mg of commercial E^ c o l i tRNA i n a 10% preparative polyacrylamide g e l . The f r a c t i o n a t i o n procedure i s given i n Materials and Methods. To face page 9 3 93 FIGURE 15 Chromatography of 100 mg of commercial E^ c o l i tRNA on DEAE-Sephadex A-50. The RNA was eluted with a 0.45-0.60 M NaCl gradient buffered i n 20 mM T r i s , pH 7.6. The flow rate was 20 ml/hr and 3.2 ml fr a c t i o n s were c o l l e c t e d . To face page 9 4 94 A260nm P P P P P !u Oi In i> O O NaCl GRADIENT FIGURE 16 Photograph of polyacrylamide gel (10%, pH 8.3 and 0.5 x 7 cm) electrophoresis patterns of the peak f r a c t i o n of each peak region of Figure 15. From l e f t to r i g h t : f r a c t i o n 90 (peak I ) , f r a c t i o n 113 (peak I I ) , f r a c t i o n 173 (peak IV) and the co n t r o l (commercial E. c o l i tRNA). To face page 95 95 polyacrylamide gel using a Canalco preparative gel e l e c t r o -phoresis apparatus as described i n Materials and Methods. The r e s u l t s are shown i n Figure 13. Aliquots from the peak f r a c t i o n s were then electrophoresed i n a 10% polyacrylamide g e l . The major peak contained 4S RNA, the second peak, 4S RNA and a trace of 5S RNA, the t h i r d peak, 4.5S RNA and the l a s t peak, 5S RNA. Figure 14 shows the f r a c t i o n a t i o n on a much larger scale where 100 mg of tRNA were used. Aliquots from the peak f r a c t i o n s were then electrophoresed i n a 10% polyacrylamide g e l . Peak I contained 4S RNA with a trace of 5S RNA, peak II contained 5S RNA, 4S RNA with trace amounts of 4.5S RNA and 5.8S RNA while peak III contained trace amounts of 4S and 5S 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) previously e q u i l i b r a t e d with 0.45 M NaCl buffered i n 20 mM T r i s , pH 7.6. The tRNA was eluted with a 0.45-0.60 M NaCl gradient (283). The gradient was checked r a d i o m e t r i c a l l y by use of a conductivity meter. The r e s u l t s are shown i n Figure 15. The o p t i c a l density pattern i s almost i d e n t i c a l to that i n Figure 13. The peak f r a c t i o n s were electrophoresed i n a 10% polyacrylamide gel and the r e s u l t s are shown i n Figure 16. Peak I contains v i r t u a l l y a l l the 4S RNA and some 4.5S RNA, peak II contains 5S RNA and some 4S RNA while peak IV contains only 5S RNA. Since the preparative gel electrophoresis gave a much better separation of 4S RNA than the DEAE-Sephadex A-50, i t FIGURE 17 Electrophoretic pattern of the low molecular weight RNA (19 40 A 2 6 0 units) from E_^  c o l i ribosomes i n a 10% preparative poly-acrylamide g e l . The f r a c t i o n a t i o n procedure i s given i n Materials and Methods. Flow rate 60 ml/hr and 3 ml f r a c t i o n s were c o l l e c t e d . S o l i d l i n e : A 2 6 o ; dotted l i n e : 1^C-amino acid acceptor a c t i v i t y . To face page 9 7 FRACTION NUMBER FIGURE 18 Photograph of polyacrylamide gel (10%, pH 8.3 and 0.5 x 7 cm) electrophoretic patterns of the peak f r a c t i o n of each peak region of Figure 17. From l e f t to r i g h t : f r a c t i o n 26 (peak I ) , f r a c t i o n 35 (peak I I ) , f r a c t i o n 70 (peak III) And the control (commercial E. c o l i tRNA). To face page 9 8 FIGURE 19 Spectrum of f r a c t i o n 26 (peak I) of Figure 17. An aliq u o t was f i r s t chromatographed on Whatman No. 1 paper and a spectrum was run on the eluted spots (A and B) as described i n the Results. To face page 99 99 100 was used to carry out the f r a c t i o n a t i o n of ribosomal RNA. Fr a c t i o n a t i o n of Ribosomal RNA By Preparative Gel E l e c t r o - phoresis The t o t a l low molecular weight ribosomal RNA was obtained as described i n Materials and Methods and separated i n a 10% preparative g e l . The r e s u l t s are shown i n Figure 17. The dotted l i n e s indicate regions of amino acid acceptor a c t i v i t y . The main absorbance peak did not correspond to the main peak of acceptance a c t i v i t y . Aliquots from the f r a c t i o n s of peak absorbance were electrophoresed i n a 10% polyacrylamide g e l . The r e s u l t s are shown i n Figure 18. The f i r s t peak contains 4S RNA, and a trace amount of 5S RNA. The second peak contains 4S RNA and some 5S RNA, and the l a s t peak contains 5S RNA only. E f f o r t s were made to characterize the main peak since i t was a region of comparatively low amino acid acceptor a c t i v i t y (a) An a l i q u o t from the main peak was spotted onto Whatman No. 1 paper and the paper chromatogram was run for 16 hours at room temperature i n a solvent system consisting of i s o b u t y r i c acid : concentrated NH<,OH:H20 (66:1:33). Two spots were obtained, one at the o r i g i n and a fluorescent spot which migrated beyond any of the standards (ADP, ATP GDP, GTP). The spots were cut out and eluted o f f the paper with H 2 O using Heppel's technique (284). A spectrum was run on the eluted spots. The r e s u l t s are shown i n Figure 19. Tracing A, the fluorescent spot does not show the t y p i c a l RNA trace while tracing B, the spot at the o r i g i n , shows a more " t y p i c a l " RNA FIGURE 20 The e f f e c t o f pH on the s p e c t r u m o f f r a c t i o n 26 (peak I ) F i g u r e 17. A i s a t pH 11.0; B a t pH 7.0; C a t pH 1.0. To f a c e page 101 101 102 trace. (b) A spectrum of the peak f r a c t i o n at various pH values was run and the r e s u l t s are shown i n Figure 20. There appears to be very l i t t l e change i n the spectrum with changes i n pH at l e a s t as f a r as the X max and X min are concerned, (c) A small amount of the peak f r a c t i o n (7.2A2 6 0 units) was put on a small DEAE-cellulose column (0.5 x 6 cm) and eluted with an (NHi») 2 C O 3 gradient. The p r i n c i p a l f r a c t i o n (2.8A260 units) was eluted i n the void volume. I t had an X max of 254 and an X min of 244. The pH of the s o l u t i o n was 9.6. A minor f r a c t i o n (0.5A2 6 0 units) was eluted with approximately 1.9 M (NHi»)2C03 but did not show a t y p i c a l RNA spectrum. I t was s i m i l a r to that of tracing B, Figure 19. There was s t i l l approximately 50% of the sample unaccounted f o r . No further sample was eluted with 2 M (NHi») 2 C O 3 i n 7 M urea, 0.1 N KOH or 1.0 N KOH. (d) Approximately 28A260 units of the peak f r a c t i o n was put on a DEAE-Sephadex A-50 column (0.9 x 32 cm) and eluted with a NaCl gradient (0.45 -1.0 M) i n 20 mM T r i s b u f f e r containing 7Murea at pH 7.4. A s i n g l e peak was eluted i n the void volume which showed an absorbance tracing 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 (c) above. Recovery was approximately 74%. The p r i n c i p a l f r a c t i o n s i n (c) and (d) were e l e c t r o -phoresed i n a 10% polyacrylamide gel but no d e f i n i t i v e r e s u l t s could be obtained. At the present moment the characterization of the main peak remains to be established. Besides contain-ing some tRNA, the p r i n c i p a l peak must contain RNA of lower FIGURE 21 Growth and pH curves of an L, c o l i pyrimidine-requiring mutant (ATCC 13135). The d e s c r i p t i o n of the growth medium i s given i n Materials and Methods. S o l i d l i n e : c e l l growth, dotted l i n e : pH. Symbols : • • growth at 4 ug/ml u r a c i l • • growth at 300 ug/ml u r a c i l • — • pH at 4 ug/ml u r a c i l • • pH at 300 ug/ml u r a c i l The arrow indicates addition of one ml u r a c i l (300 ug/ml). To face page 10 3 1 0 3 10 4 molecular weight since presumably t h i s would be eluted f i r s t from the preparative g e l . This low molecular weight RNA may jus t be degraded RNA and the presence of such a high concen-t r a t i o n of t h i s material may be due to the nature of the preparative gel procedure. This may be one of the p i t f a l l s of the procedure and t h i s technique was abandoned i n favor of the demonstrated s u p e r i o r i t y of Sephadex G-100 as a means of fr a c t i o n a t i n g low molecular weight RNA. Exchange Experiments An E^ c o l i pyrimidine-requiring mutant (ATCC 13135) was grown i n a minimal-salts medium i n the presence of 4 ug urac i l / m l as described i n Materials and Methods. Figure 21 shows the growth curve of the mutant at two d i f f e r e n t u r a c i l concentrations. When the c e l l s were grown i n the lower concentration of 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 . Addition of the higher concentration of u r a c i l resulted i n an immediate marked increase i n c e l l growth. When the c e l l s were grown i n the higher concentration of u r a c i l , c e l l growth also proceeded normally and then l e v e l l e d o f f . The addition of the same concentration of u r a c i l had no further e f f e c t on c e l l growth. The pH of the medium decreased during c e l l growth but remained constant during the stationary periods regardless of the concentration of u r a c i l i n the medium. Once the growth pattern of t h i s mutant s t r a i n was esta-FIGURE 22 Displacement of l a b e l l e d tRNA bound to E^ _ c o l i ribosomes. The E. c o l i mutant described i n Figure 21 was used. The preparation of the ribosomes i s described i n Materials and Methods. Symbols : Tube 1 • • stripped tRNA + ATA Tube 2 A — A aa-tRNA + PM Tube 3 • • stripped tRNA + ATA + PM Tube 4 * d aa-tRNA + ATA + PM Conditions of displacement: At 0 wash each tube contained i n a t o t a l volume of 10 ml, 2 mg of the p a r t i c u l a r unlabelled tRNA and 150A260 units WRib buffered i n 10 mM T r i s , 10 mM Mg(OAc) 2, pH 7.6. Tubes 1, 3 and 4 were made 70 uM with respect to ATA and tubes 2, 3, and 4 ImM with respect to PM. . Tubes 1 and 2 were incubated f o r 30 mins at 0° and tubes 3 and 4 for 30 mins at 30°*. A f t e r the second wash each tube was incubated at 30° i n the presence of 10 mg of unlabelled tRNA. A f t e r the fourth wash each tube was incubated at 24° f o r 20 mins i n a so l u t i o n containing 0.5 mM GTP, 3 mM ATP, 50 mM NH^Cl, 750yl S100, 400ul S100A and 10 mg of unlabelled tRNA. Aft e r the f i f t h wash each tube was incubated at 24° for 20 mins i n a buffe r containing 50 mM T r i s , 5 mM Mg(OAc )2/ pH 7.6 and 10 mg of unlabelled tRNA. FIGURE 22 - continued After the s i x t h wash each tube was incubated at 37° for 30 mins i n a so l u t i o n containing 50 mM KC1 and 10 mg of unlabelled tRNA. A f t e r the seventh wash each tube was incubated at 0° with 0.1 mM Mg(OAc) 2 buffered at pH 7.3 (buffer K) and 10 mg of unlabelled tRNA. * A f t e r each incubation period, the WRib obtained by c e n t r i -fugation were resuspended i n 10 ml of solut i o n containing the p a r t i c u l a r components for displacement. To face page 105 105 106 b l i s h e d the c e l l s were grown i n the same minimal-salts medium but t h i s time 8 ug u r a c i l / m l of medium was added. One m i l l i -curie of u r a c i l - 6 - 3 H (9.16 ug) was also added to l a b e l the c e l l s . The c e l l s were grown to the late log phase and then harvested. Ribosomes were prepared i n the usual manner and were found to be very active i n a protein-synthesizing system (Table I ) . A preliminary experiment was c a r r i e d out to study the conditions necessary to displace 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 Figure 22. Displacement appears to be v i r t u a l l y complete a f t e r s i x washes regardless of the conditions used. I t was decided to carry out an exchange experiment i n the same manner as was done by Cannon et al. (148) . In t h i s experiment, a buffer system i s employed under c e r t a i n condi-tions which enables exchange between unlabelled tRNA and l a b e l l e d ribosomal bound tRNA to r e a d i l y take place. The 3H-WRib were mixed i n a buffer containing 10 mM T r i s , 10 mM Mg + + at pH 7.4. Unlabelled c o l i B tRNA (General Biochemicals) was added (15x the amount of l a b e l l e d tRNA calculat e d to be present on the WRib) and the mixture was incubated at 0-4° for 30 mins with occasional s t i r r i n g . A f t e r the incubation period the mixture was centrifuged at 105,000 x g f o r 5 hrs. The p e l l e t was mixed with fresh b u f f e r , an a l i q u o t was removed to determine the amount of exchange that had taken place and then unlabelled tRNA was added and the incubation repeated. The washings were repeated u n t i l 10 7 TABLE V Exchange of l a b e l l e d tRNA bound to E. c o l i ribosomes with unlabelled tRNA No. of washings Ribosomal p e l l e t * * Non-pelleted ribosomes* (cpm) (cpm) 0 33884 1 25809 57187 2 23548 4663 3 18333 31026 4 15080 16968 5 14974 3579 6 13728 1047 * The non-pelleted ribosomes were put through a M i l l i p o r e f i l t e r (1.2 y ). Ribosomes with bound tRNA remained on the f i l t e r (290). The f i l t e r was dried and counted i n a toluene s c i n t i l l a t i o n mix. ** The ribosomal p e l l e t was mixed with 10 ml of buffe r (see Text) and a 5 y l a l i q u o t was counted i n a dioxane s c i n t i l l a -t i o n mix. FIGURE 23 Exchange of l a b e l l e d tRNA bound to E^ c o l i ribosomes with unlabelled tRNA. Ribosomes were prepared as mentioned i n Figure 22 from the E^ c o l i mutant described i n Figure 21. De t a i l s of the exchange conditions are given i n the Results. To face page 10 8 10 8 N U M B E R O F W A S H I N G S i 109 exchange was complete (Table V and Figure 23). According to the table a great number of counts were found i n the super-natant (non-pelleted ribosomes). The extremely high l e v e l s of counts i n some of the supernatants was probably due to the presence of some ribosomes r e s u l t i n g from incomplete ce n t r i f u g a t i o n . Although exchange appeared to be v i r t u a l l y complete a f t e r four washes, two more exchanges were c a r r i e d out u n t i l the loss of counts to the supernatant was minimal. This minimal loss was achieved by centrifuging the ribosomal mixture for extended periods of time to ensure that a l l of the ribosomes had been p e l l e t e d . These ribosomes were found to be i n a c t i v e i n a protein-synthesizing system. Following the exchange experiment, the t o t a l ribosomal RNA was prepared from these 3H-WRib as described i n Materials and Methods. This RNA was dissolved i n 10 mM Mg(OAc)2 buffered at pH 7.6 (buffer F) which was then made two molar with respect to NaCl. The high molecular weight RNA p r e c i p i t a -ted immediately and was c o l l e c t e d by centrifugation while the low molecular weight RNA remained i n the supernatant. The high molecular weight RNA was redissolved i n buffer F and r e p r e c i p i t a t e d i n 2 M NaCl. The supernatant was combined with the previous one. Sodium acetate (1.5 M, pH 5.2) was added to give a 2% s o l u t i o n and the low molecular weight RNA was subsequently p r e c i p i t a t e d by the addition of 3 volumes of cold ethanol. This low molecular weight RNA was put on a Sephadex G-100 column (200 x 3 cm) and eluted with 50 mM NaCl. FIGURE 24 E l u t i o n pattern from Sephadex G-100 of the low molecular weight RNA (2000A2 6 0 units) bound to l a b e l l e d c o l i ribosomes a f t e r exchange with unlabelled tRNA. The ribosomes and the exchange conditions are described i n Figure 23. The RNA was fractionated on a 3 x 200 cm column and eluted with 50 mM NaCl at a flow rate of 6 ml/hr. Five ml fr a c t i o n s were c o l l e c t e d . To face page 110 FRACTION NUMBER FIGURE 25 Photograph of polyacrylamide gel (10%, pH 8.3 and 0.5 x 7 cm) electrophoresis patterns of various f r a c t i o n s from Figure 24. From l e f t to r i g h t : f r a c t i o n s 85, 106, 116, 121, 125, 130, 135, 142 and the control (commercial E. c o l i tRNA). To face page 111 I l l 112 TABLE VI D i s t r i b u t i o n of l a b e l l e d 3H-RNA i n E^ c o l i ribosomes a f t e r exchange with unlabelled tRNA Fra c t i o n cpm/A 26o u n i t high molecular weight RNA 19465 5S RNA 2707 4S RNA 55 113 The r e s u l t s are shown i n Figure 24. The f i r s t peak contained high molecular weight RNA while the re s t contain the major part of the RNA as indicated. Aliquots of the peak f r a c t i o n s and a number of fr a c t i o n s between the second and t h i r d peaks were electrophoresed i n 10% polyacrylamide gels. The re s u l t s are shown i n Figure 25. The f i r s t peak ( f r a c t i o n 85) also contains a small amount of 5.8S and 5S RNA. The second peak ( f r a c t i o n 116) contains only a trace of 4S RNA while the t h i r d peak ( f r a c t i o n 142) contains v i r t u a l l y a l l the 4S RNA and some 4.5S RNA. The A 2 6 0 r a t i o of 4S : 5S RNA had increased to almost 3.5 i n ribosome exchanged with unlabelled tRNA. This i s comparable to about 5.7 molecules of 4S RNA to one molecule of 5S RNA*. In untreated ribosomes the r a t i o of 4S : 5S RNA on a t o t a l RNA basis varied from 0.58 (Table IV) to 0.90 (Table I I ) . This amounted to 1-2 molecules of tRNA bound per molecule of 5S RNA; a value which compared very favourably with the l i t e r a t u r e (286-289). Aliquots of the 3 peaks fr a c t i o n s i n Figure 24 were taken and the amount of l a b e l l e d 3H-RNA was determined. The re s u l t s are shown i n Table VI. I t appears that almost 100% exchange had taken place between unlabelled tRNA and the l a b e l l e d tRNA o r i g i n a l l y bound to 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 103 and 2.5 x 103 respectively. FIGURE 26 Chromatography on BD-cellulose of 4S RNA (140A 26o units) bound to E_^  c o l i ribosomes a f t e r exchange with unlabelled tRNA. Fractions 135-170 (Figure 24) were fractionated on a column (100 x 0.9 cm) with the indicated (dashed line) gradient of NaCl containing 10 mM MgCl 2 i n a t o t a l volume of one l i t e r . The flow rate was 42 ml/hr and 5 ml fracti o n s were c o l l e c t e d . At the indicated point, e l u t i o n was continued with a 0-30% ethanol gradient i n 1 M NaCl containing 10 mM MgCl 2 i n a t o t a l volume of 400 ml. The flow rate 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 . To face page 114 114 MOLARITY OF NaCl FIGURE 27 Chromatography on BD-cellulose of 4S RNA (140A 26 0 units) normally bound to E^ c o l i ribosomes. Fractions 126-140 (Figure 11) were fractionated on a column (112 x 0.9 cm) with the indicated (dashed line) gradient of NaCl containing 10 mM MgCl 2 i n a t o t a l volume of one l i t e r . The flow rate 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 . At the indicated point, e l u t i o n was continued with a 0-30% ethanol gradient i n 1 M NaCl containing 10 mM MgCl 2 i n a t o t a l volume of 400 ml. The flow rate was 42 ml/hr and 5 ml fr a c t i o n s were c o l l e c t e d . To face page 115 116 The acrylamide gel analysis (Figure 25) of various f r a c t i o n s from the Sephadex G-100 column (Figure 24) indicated that f r a c t i o n s 135-170 contained v i r t u a l l y a l l the 4S RNA and some 4.5S RNA but were devoid of 5S RNA. These f r a c t i o n s were put on a BD-cellulose column (100 x 0.9 cm) previously e q u i l i b r a t e d with 0.35 M NaCl i n the presence of 10 mM Mg + +. The RNA was eluted with a (0.35-1.0 M) NaCl gradient i n a t o t a l volume of one l i t e r . This was followed by a 0-30% ethanol gradient i n 1 M NaCl and 10 mM MgCl2. To t a l volume of t h i s gradient was 400 ml. The r e s u l t s are shown i n Figure 26. Four peak regions were i s o l a t e d i n the NaCl gradient although the second peak region i s i t s e l f a c t u a l l y made up of 3 peaks. The ethanol gradient contains one d e f i n i t e peak and a shoulder which may a c t u a l l y be a second component. Figure 27 shows the pattern of low molecular weight RNA normally bound to E^ c o l i ribosomes. I t i s somewhat d i f f e r e n t from the pattern shown i n Figure 26. In place of the f i r s t peak (Figure 26) there i s a shoulder. There i s a sharp, well-defined second peak and a broader t h i r d peak which shows a small peak on i t s t r a i l i n g edge. The peaks i n the ethanol gradient were much better separated than i n the previous experiment (Figure 26). Figure 28 shows the o p t i c a l density pattern obtained when commercial Ej_ c o l i tRNA was fractionated on BD-cellulose. The s a l t gradient eluted two d e f i n i t e peaks, the second having a s l i g h t shoulder which may indicate the p o s s i b i l i t y of a t h i r d component. The ethanol f r a c t i o n contained only one component FIGURE 28 Chromatography on B D - c e l l u l o s e o f c o m m e r c i a l E ^ c o l i tRNA. The RNA ( 3 6 5 0 A 2 6 o u n i t s ) was f r a c t i o n a t e d on a column (106 x 1.5 cm) w i t h t h e i n d i c a t e d (dashed l i n e ) g r a d i e n t o f N a C l c o n t a i n i n g 10 mM M g C l 2 i n a t o t a l volume o f t h r e e l i t e r s . The f l o w r a t e was 90 m l / h r and 20 ml f r a c t i o n s were c o l l e c t e d . A t 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 c o n t i n u e d w i t h 1.10 N a C l c o n t a i n i n g 10 mM M g C l 2 i n 10% (v/v) e t h a n o l . To f a c e page 117 FRACTION NUMBER 118 but this may have been due to the fact that a gradient was not employed. Aliquots were taken from every second fraction (Figure 26) to see i f the labelled tRNA that remained bound to c o l i ribosomes after the exchange with unlabelled tRNA was spread evenly throughout the elution pattern, or whether i t was concentrated in a certain region. It was found to be spread uniformly throughout the fractions eluted with the NaCl gradient. The radioactivity amounted to 25-50 cpm per A 2 6 0 unit. In the ethanol gradient the radioactivity reached a maximum level of 250 cpm per A 2 6 0 unit. An aliquot of the peak fraction was electrophoresed in a 10% polyacrylamide gel and was found to contain some 4.5S RNA. This may account for the high counts in this particular fraction. Another possibility i s that according to Table III,tRNA T r p is bound to WRib in the highest amount and therefore i t may not be as readily exchanged as the other tRNAs. Since tRNATr^ is found solely in the ethanol fraction (295) i t therefore could account for the high counts. The former explanation seems to be more plausible since there i s no reason to believe that one pa r t i -cular tRNA is less vulnerable to exchange than another. 119 DISCUSSION The experiments described i n the previous sections were c a r r i e d out i n an attempt to c l a r i f y the b i o l o g i c a l role of the low molecular weight RNAs associated with E_j_ c o l i r i b o -somes. In p a r t i c u l a r , these experiments were designed to characterize the RNA bound to c o l i ribosomes which had undergone extensive washing procedures. Some of these RNAs were c l e a r l y i d e n t i f i e d but i n most cases t h e i r function could not be established. Nevertheless, a method was found for preparing ribosomes devoid of a l l 4S components but no evidence was obtained f o r a s p e c i f i c 4S f r a c t i o n which might function during protein synthesis i n the chain termination mechanism. This r e s u l t helps to confirm recent reports- on the non-existence of a chain-terminating tRNA. The mechanism of chain termination i n protein biosynthesis i s s t i l l an unsolved problem. Present evidence suggests that there may be three nonsense or terminating codons, UAA, UAG and UGA. According to the Wobble Hypothesis (30 7), however, UGA may also code fo r Cys and Trp. No suppressor has yet been found which suppresses only ochre mutants (UAA), although suppres-sors e x i s t which suppress both ochre and amber mutants (UA 0). Since UAA has been found to be the terminator codon f o r a l l the phage proteins thus f a r i d e n t i f i e d , i t i s possible that at l e a s t one s p e c i f i c terminating tRNA could be involved. However, the groups i n v e s t i g a t i n g t h i s problem could not demonstrate such a tRNA species i n the termination step. Most of the information about the mechanism of chain 120 t e r m i n a t i o n was obtained by the use of two assay systems.. In one of these, Capecchi (43, 75) used as mRNA, the RNA from a mutant R17 phage, i n which the seventh codon i n the coat p r o t e i n gene was a nonsense codon (UAG). The formation of the hexapeptidyl-tRNA was c a r r i e d out as d e s c r i b e d pre-v i o u s l y (p. 29). The r e s u l t i n g hexapeptidyl-tRNA remained attached t o the mRNA-ribosome complex. The r e l e a s e of f r e e hexapeptide from t h i s complex depended on a p r o t e i n component, designated r e l e a s e f a c t o r (R f a c t o r ) from the high speed supernatant (S100) of c o l i . B r e t s c h e r (84) , u s i n g the same assay system, incubated the S100 supernatant under m i l d a l k a l i n e c o n d i t i o n s and subsequently t r e a t e d i t w i t h p e r i o -date t o destroy the amino a c i d acceptor a b i l i t i e s of the endogenous tRNAs. He then added only those aa-tRNA species needed to form the hexapeptide plus the p e r i o d a t e - t r e a t e d supernatant and got c h a i n - t e r m i n a t i o n . Nirenberg's group (88), using a AUG•UAA•ribosome complex as the t e r m i n a t i o n assay, was able t o get chain t e r m i n a t i o n t o take p l a c e upon a d d i t i o n of the crude R f a c t o r . They subsequently found t h a t the R f a c t o r was a c t u a l l y two d i f f e r -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, 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 a l s o i s o l a t e d (90, 92). Recently I s h i t s u k a and K a j i (109) 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 suggested worked hand-in-hand w i t h the R f a c t o r - - t h e R f a c t o r hydrolyzed the e s t e r l i n k between the peptide 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 of the groups working i n th i s f i e l d s t i p u l a t e d i n t h e i r discussions that t h e i r results were not conclusive evidence for the nonexistence of a chain-terminating tRNA. In contrast to the case of 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 involved, the requirement f o r a s p e c i f i c tRNA i n termination has not, as yet, been shown but the present understanding of the problem i s that such a tRNA i s not required. The chain-terminating experiments outlined above i n which highly p u r i f i e d tRNAs were used (84, 88), neverthe-less were not properly con t r o l l e d since i t can be argued that the terminating tRNA could have remained bound to the ribo-somes. These previous investigators f a i l e d to show that t h e i r ribosomes or ribosomal subunits were devoid of 4S RNA. This tRNA could 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 or tRNA-l i k e component could be an i n t e g r a l part of the ribosome. Previous investigators studied the proteins involved i n the termination mechanism and from t h e i r results concluded that the possible involvement of a s p e c i f i c RNA component was remote. This report i s the f i r s t known d i r e c t study of the RNA bound to ribosomes with respect to i t s possible involvement i n chain-termination. The following questions which have yet to be posed concerning chain-termination could be asked: (1) What i s the nature of the low molecular weight RNA associated with p u r i f i e d ribosomes? 122 (2) Is there a s p e c i f i c low molecular weight RNA other than 5S RNA which cannot be e q u i l i b r a t e d with tRNA? (3) I f such an RNA exists i s i t involved i n chain term-ination? In order to begin to answer the questions i t was neces-sary to obtain a clean ribosome preparation which was active i n protein synthesis. Ribosomes from c o l i were chosen fo r the study for three reasons: (a) they are w e l l character-i z e d , (b) the ease with which active ribosomes could be obtained, and (c) a l l previous chain-termination studies had been c a r r i e d out with these ribosomes. The l a t t e r reason (c) was of p a r t i c u l a r importance since i t would be easier to c o r r e l a t e the r e s u l t s obtained i n these studies with those reported i n the l i t e r a t u r e . At the beginning of the project Nirenberg's group had reported the best ribosome preparation (WRib) and t h e i r procedure was followed. The ribosomes used i n a l l experiments to be discussed were r e a d i l y prepared from c o l i B c e l l s grown to the mid-log or l a t e log phase (see Materials and Methods). The preparation involved seven incubations with 0.5-1.0 M NHi»Cl at various concentrations of magnesium buffered at pH 7.4. These steps removed a l l enzymes (and RNA) not t i g h t l y bound to ribosomes. A number of investigators had also shown that prolonged washing with 0.5 M NHJ.C1 deactivated or removed ribosomal RNase I (29 6-29 8) . Others have found v a r i a b l e results i n that some of the RNase a c t i v i t y had been removed by NHi»Cl washing but the ribosomes s t i l l retained a s i g n i f i -cant l e v e l of RNase (29 8) . Ribosomal a c t i v i t y was determined 123 by following the uptake of l l ,C-Phe i n a polyU-dependent phenylalanine incorporation system. The a c t i v i t y of the pre-parations varied between 50-100 f o l d over the c o n t r o l or back-ground l e v e l (see Results, p. 68). The ribosomes were 67% RNA as contrasted with the usual value of 60-6 3% (6) which was a d d i t i o n a l confirmation of the high purity obtained. The choice 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 to which the ribosomes are to be put, the nature of the impurities which are to be removed, etc* Often a balance must be found between the p u r i f i c a t i o n and over-handling of the ribosomes since handling may destroy t h e i r b i o -l o g i c a l a c t i v i t y . In general, there i s no absolute standard of p u r i t y . Each preparation must be judged by an operational c r i t e r i o n . Keeping the l a t t e r statement i n mind, a development occurred at a stage when the project was already considerably advanced. Iwasaki et a l . (219) reported a new and very quick method of obtaining very active ribosome preparations. The method e s s e n t i a l l y involved washings i n 1 M NH..C1 followed by e l u t i o n through a DEAE-cellulose column (RSI, Chart I I ) . The a c t i v i t y of these fresh ribosome preparations was approximately 150-fold over the control or background l e v e l . Because of the advanced stage of the present project, t h i s l a t t e r method of preparing ribosomes was not adopted since i t would have been necessary to repeat a l l the experiments already completed. For th i s reason and those already mentioned, the ribosome prepara-t i o n i n use was deemed s u i t a b l e f o r the experiments i n which 124 i t would be employed. The a c t i v i t y of the ribosome preparations was quite variable as shown in Table I. Perhaps the difference i n a c t i v i t y l e v e l s was caused by variations i n handling during the work-up of the ribosomal preparation. I t i s w e l l known that ribosomes which are quite stable at low temperature and i o n i c strength become unstable when ei t h e r the temperature or i o n i c strength i s raised or the concentration of Mg + +-ions i s reduced. Under the former conditions, the ribosomes were found by numerous investigators to have latent ribosomal RNase I a c t i v i t y while the l a t t e r conditions tended to activate the enzyme (298). Following such treatments which disrupt the structure of the ribosome, the enzyme, i f present, i s able to attack both the ribosomal RNA and added free RNA under condi-tions of temperature, i o n i c strength and Mg + +-ion concentration where i n t a c t ribosomes showed no RNase a c t i v i t y (298). The ribosomal preparations used i n Table I (RSI, II and III) were two month o l d WRib which had been stored at -70°. The o r i g i n a l fresh preparation had an a c t i v i t y 82-fold greater than the blank which contained no ribosomes (Table I ) . Most investigators have in d i c a t e d that t h e i r ribosomal preparations remained active for up to three weeks (219) while others have obtained active preparations with s i x month o l d preparations (218). Although Nirenberg (218) was able to freeze and thaw his WRib preparations several times without undue loss of a c t i v i t y , the WRib preparation used i n experiment I I , Table I was almost i n a c t i v e a f t e r freezing and thawing twice. This 125 may have been due t o the f a c t t h a t the p r e p a r a t i o n was already two months o l d . The reason f o r our i n a b i l i t y t o maintain a c t i v e preparations over extended periods of time may a l s o have been the r e s u l t of the method used to f r e e z e the prepara-t i o n s . They were q u i c k l y f r o z e n i n dry i c e p r i o r t o storage at -70°. On the other hand Nirenberg f r o z e h i s e x t r a c t s i n l i q u i d N 2 p r i o r to storage i n l i q u i d N 2 r e f r i g e r a t o r s . Even though the preparations were kept at -70° the p o s s i b l e presence of some nuclease a c t i v i t y cannot be discounted. For i n s t a n c e , Szer (299) , using an RNase I ~ s t r a i n of E^ c o l i , found almost complete disappearance of 23S RNA w i t h the concomitant forma-t i o n of 16S RNA i n the l a r g e r ribosomal subunit at 0°. The f i n a l products suggested the involvement of an RNase IV found i n the RNase I " s t r a i n . RNase I I , which i s a l s o present i n t h i s s t r a i n , loses 9 0% of i t s a c t i v i t y a f t e r 24 hrs when kept c o l d or frozen (300). However, when our " o l d " WRib prepara-t i o n s were passed through a DE-22 column and e l u t e d w i t h 1 M NHi»Cl, the o r i g i n a l a c t i v i t y of the WRib, 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, 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 reported by Scheps e t a l . (26 8) who observed t h a t the a c t i v i t y of a ribosomal e x t r a c t c o u l d be r e s t o r e d by a temperature dependent p r e i n c u b a t i o n i n the presence of 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 suggest t h a t t h i s i s due to a decrease i n the amount of 70S ribosomes which were found t o have i n c r e a s e d i n the formerly 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 the de f e c t i n v o l v e d both subunits and was not due t o an i n a c t i v e 126 S100 which contains the synthetase a c t i v i t y . I t should also be borne i n mind that as mentioned e a r l i e r , washing ribosomes with 0.5 M NH4CI tends to remove some of the ribosomal bound RNase I (296-298). But Ochoa's group (219) found that a f t e r two prolonged washings with 0.5 M NH^Cl the ribosomes s t i l l contained a high l e v e l of RNase a c t i v i t y . However, when the ribosomes were chromatographed on a DEAE-cellulose column the RNase a c t i v i t y was reduced by about 9 9%. These ribosomes were active i n protein synthesis. This may account for the very active WRib preparations which were obtained when the WRib were prepared by washing through a DE-22 column (RSI, Chart I I , Table I ) . The reason for not using t h i s preparation i n a l l experiments was already discussed. These observations show that RNase can be removed from E^ c o l i ribosomes without caus-ing t h e i r i n a c t i v a t i o n ; the enzyme normally appears, however, to be bound very firmly to the ribosomes. Unfortunately, none of the fresh ribosomal preparations used i n the present studies was checked f o r the presence of RNase a c t i v i t y , and although they were extensively treated with 1 M NHi»Cl they were not subsequently eluted through a DEAE-cellulose column. I t may be reasonable to assume that since these RNases are normally so t i g h t l y bound to the ribosomes, at l e a s t some RNase a c t i v i t y remained associated with them (WRib). I t i s also known that as a b a c t e r i a l culture enters the l a t e logarithmic and stationary phases i t s a b i l i t y to y i e l d an extract active i n synthesizing peptides decreases. This has been observed f o r several b a c t e r i a l species (218, 266, 267) 127 and i s seen as w e l l with natural mRNA and i n the polyU-directed synthesis of polyphenylalanine. To compare the a c t i v i t y of c e l l - f r e e protein-synthesizing extracts from d i f f e r e n t lots of c e l l s , the c e l l s should be harvested i n the same p h y s i o l o g i c a l s t a t e , a condition which i s often d i f f i c u l t to obtain (experi-ment I I , Table I ) . The c e l l s used i n a l l the experiments except the exchange studies were obtained commercially i n two batches. One batch was harvested at the mid-log phase while the other was harvested at the l a t e log phase. The exchange experiments were c a r r i e d out with 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 harvested i n the l a t e log to stationary phase. Although WRib prepared from each batch of c e l l s were very active i n protein synthesis, the a c t i v i t y per A 2 6 0 u n i t v a r i e d from batch to batch and t h i s may have been due, i n part, to the stage at which the c e l l s had been harvested. Figure 1 shows the separation of low molecular weight RNA from the d i f f e r e n t ribosome preparations (Chart I I , Table I) a f t e r electrophoresis i n a 10% polyacrylamide g e l . There appears to be a marked diffe r e n c e i n the amount of tRNA bound to ribosomes which have been put through a DEAE-cellulose column and subsequently eluted with 1 M NH^Cl. I t appears quite l i k e l y that the column has not only removed ribosomal aggregates but also loosely bound tRNA which may block s i t e s on the ribosomes, and/or a l t e r the conformation of the ribosome i n such a way as to prevent the normal tRNA exchange from taking place. Once an active ribosome preparation was obtained experi-128 ments were undertaken to prepare WRib devoid of a l l 4S com-ponents while at the same time maintaining the a c t i v i t y of the ribosome. Various methods were used to remove bound tRNA from ribosomes. The results shown i n Figure 2 indicated that puro-mycin (PM) treatment alone d i d not remove a l l the bound tRNA. This was expected since PM occupies only one s i t e on the ribo-some, the peptidyl s i t e . Aminoacyl-tRNA bound to the acceptor s i t e does not react with PM ( 2 6 9 , 285) . K u r i k i and K a j i (285) have shown that while PM reduced 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. In the absence of soluble enzymes and GTP, the i s o l a t e d complex of ribosomes, tRNA and peptidyl-tRNA bound a d d i t i o n a l tRNA suggesting that the ribosome contained two s i t e s f o r aminoacyl-tRNA and one s i t e f o r peptidyl-tRNA. These data are consistent with the hypothesis that during polypeptide synthesis the s i t e for peptidyl-tRNA and one s i t e for aminoacyl-tRNA are constantly occupied but the other s i t e f o r aminoacyl-tRNA i s occupied t r a n s i e n t l y . Only i n the absence of peptide bond formation are both s i t e s for aminoacyl-tRNA constantly occupied. At any one time, 50% of the t o t a l bound tRNA should be displaced by PM and t h i s would account f o r the decreased amount of bound tRNA observed i n Figure 2 . These PM-treated WRib were also found to be more active than the o r i g i n a l preparation; a r e s u l t that i s i n agreement with that observed by Scheps et a l . (26 8 ) . They suggested that the i n a b i l i t y of the ribosome to respond to polyU was due to t h e i r being blocked by unfinished polypep-t i d e chains. PM overcame this by d i s p l a c i n g the peptidyl-tRNA, 129 causing the release of growing peptide chains. D i a l y s i s of PM-treated WRib against low Mg + + removed a l l the bound tRNA (Figure 2). This was to be expected since the removal of the nascent polypeptide chain by PM would remove the s t a b i l i z i n g e f f e c t i t exerted on the binding of the tRNA to the ribosome. Under these conditions the ribosomes would be converted to t h e i r subunits and i t has been found by some investigators that ribosomal subunits are devoid of 4S RNA material (265, 270, 271). Other investigators have suggested that ribosomal subunits are only devoid of 4S RNA material when treated f i r s t with PM but i n the absence of PM the 50S ribosomal subunit contains peptidyl-tRNA (115, 272). Unfor-tunately the d i a l y s i s against low Mg + + l e f t the WRib v i r t u a l l y i n a c t i v e . This may have been due to the f a c t that the decreased Mg'*"'" concentration d e s t a b i l i z e d the nucleoprotein structure r e s u l t i n g i n the a c t i v a t i o n of the late n t ribosomal RNase I (29 8) . D i a l y s i s of the WRib against low Mg + + ion concentration (0.1 mM) removed v i r t u a l l y a l l the bound tRNA (Figure 3). Cannon et a l . (148) have found that i n the absence of protein synthesis or mRNA, and at 0.1 mM Mg + + ion concentration, the ribosomes were di s s o c i a t e d i n t o t h e i r subunits and i n the ensuing process a l l the bound tRNA was shown to be completely washed o f f . A f t e r protein synthesis i n a c e l l - f r e e extract of E. c o l i , a small f r a c t i o n of the tRNA that was bound to the ribosomes i n high Mg + + ion concentration became r e s i s t a n t to being washed o f f i n low Mg + + ion concentrations. According to 130 Cannon e t a l . t h i s amounted to about h a l f a molecule of tRNA per ribosome. This had been i n t e r p r e t e d as due to the presence of a nascent po l y p e p t i d e c h a i n on the tRNA which s t a b i l i z e d the b i n d i n g of the tRNA t o the ribosome. As was found i n the previous experiments w i t h PM, d i a l y s i s against low magnesium not only removed a l l 4S RNA but a l s o l e f t the ribosomes v i r t u a l l y i n a c t i v e . Once agai n , the reason f o r the i n a c t i v e p r e parations may have been due t o nuclease a c t i o n . Ribosomal subunits were prepared from these d i a l y z e d ribosomes by c e n t r i f u g a t i o n through a sucrose g r a d i e n t . The r e s u l t s (Figure 5) showed t h a t there was v i r t u a l l y no 4S RNA m a t e r i a l bound to the 50S ribosomal s u b u n i t and a b s o l u t e l y none bound t o the 30S ribosomal s u b u n i t . Although the subse-quent C s C l treatment of these subunits should have removed only the s p l i t p r o t e i n s l e a v i n g behind the 23S and 40S ribosomal subunits (60), i t appears t h a t some degradation must have occurred to account f o r the a d d i t i o n a l bands observed i n Fig u r e 5; f o r example, a 5S RNA band i n the 30S p a r t i c l e and a 4S RNA band i n the 50S p a r t i c l e . I t has been shown t h a t i n the absence of mRNA, 30S. p a r t i c l e s do not 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 the 50S ribosomal s u b u n i t s . In the presence of messenger there 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 th a t tRNA-linked nascent p o l y p h e n y l a l a n i n e remains attached 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 of the ribosomes i n t o subunits (263). E l s e n (264, 265), on the other hand, observed the r e l e a s e of 4S RNA m a t e r i a l from the 50S p a r t i c l e i n the presence 131 of high s a l t . Attempts were made to obtain an active ribosomal prepara-t i o n using the i n d i v i d u a l subunits instead of whole WRib. The subunits, instead of WRib, were used i n the assay system f o r determining polyphenylalanine synthesis as described i n Materials and Methods. Twice as much of the 50S ribosomal subunit was added to the system as 30S ribosomal subunit be-cause of the differences i n molecular weight (9 3) . The ribosomal a c t i v i t y was found to be just above the blank which contained no ribosomes. The following reasons may account f o r the f a i l u r e to achieve ribosomal a c t i v i t y : (a) the subunits were prepared from 2-month old WRib and this together with variati o n s i n handling during the work-up and preparation of subunits may have i n a c t i v a t e d the preparation, (b) the prepara-t i o n of ribosomal subunits involved d i a l y s i s against low magnesium which i n turn causes the d e s t a b i l i z a t i o n of the nucleoprotein structure r e s u l t i n g i n the a c t i v a t i o n of the latent ribosomal RNase I a c t i v i t y . This RNase has been found to be located exclusively on the 30S subunit (298). However, i t has been estimated that no more than one ribosome i n about ten would carry a molecule of RNase (29 8). Szer (299) found that i f f r e s h l y i s o l a t e d 70S ribosomes from an RNase I" s t r a i n of L c o l i are fractionated i n t o subunits and kept at 0°, the degradation goes further and both 23S and 16S RNAs are halved. The f i n a l products suggested the involvement of an RNase IV found i n this RNase I" s t r a i n . The e f f e c t of RNases on whole WRib as compared to t h e i r subunits w i l l be discussed l a t e r , 132 (c) the conditions used to observe ribosomal subunit a c t i v i t y were not optimal. For instance, the r a t i o of Mg + +/ATP w i l l determine the degree of attachment of the amino acids to the tRNAs (27 8). The r a t i o which i s optimal i n a system containing WRib may not be optimal i n the same system containing the sub-units instead of WRib. According to Pestka and Nirenberg (159) the a c t i v i t y of d i f f e r e n t 30S ribosomal subunit prepara-tions varied and 70S ribosomes formed by r e a s s o c i a t i o n of p u r i f i e d 30S and 50S ribosomal subunits were only about h a l f as active i n binding aa-tRNA as non-dissociated 70S ribosomes. I t should be noted here that a f t e r the above experiments had been completed Tompkins et a l . (93) prepared subunits from ribosomes prepared i n an i d e n t i c a l manner as i n the present work and with these subunits, they were able to obtain an active ribosomal preparation. This preparation was also active i n chain termination using the Caskey termination assay (88). Since our subunits contained n e g l i g i b l e amounts of tRNA and since t h i s termination assay contained only i n i t i a t o r tRNA (fMet-tRNA) i t may be assumed, i n d i r e c t l y , that the chain-termination mechanism does not require a s p e c i f i c terminating tRNA. This point w i l l be c l a r i f i e d further on i n the t h e s i s . At this stage i n the development of the thesis the following points had been established: (a) a method f o r pre-paring active ribosomes , (b) treatment of WRib with PM removed some of the bound tRNA leaving the preparation more active than the o r i g i n a l , and (c) d i a l y s i s against low magnesium dis s o c i a t e d the ribosomes into subunits devoid of 4S RNA and i n a c t i v e i n 133 protein synthesis. Nirenberg was able to get chain-termination using whole ribosomes and since active ribosomes could be r e a d i l y prepared i t was f e l t that the proposed studies of the low molecular weight RNA, p a r t i c u l a r l y the tRNA bound to ribosomes, could be c a r r i e d out with these preparations. As mentioned e a r l i e r the WRib used i n a l l the experiments may have contained some nucleases as evidenced by the f a c t that ribosome a c t i v i t y could be restored by e l u t i o n through a DEAE-cellulose column which i s known to remove RNases (281). The same e f f e c t could have been due to the removal of ribosomal aggregates or tRNA which blocked s i t e s on the ribosome and i n so doing prevented the normal tRNA exchange from taking place. Previous evidence suggested that nuclease a c t i v i t y on WRib remained la t e n t provided the v a r i a t i o n s i n handling during the preparation of WRib were minimized and therefore this was the only precaution taken to eliminate nuclease a c t i v i t y . Since experiments were c a r r i e d out only with active preparations, con t r o l experiments to determine the nuclease a c t i v i t y of these preparations were considered unnecessary. Most of the p e r t i -nent data recorded i n the l i t e r a t u r e also tended to suggest that tRNA bound to whole ribosomes was RNase r e s i s t a n t . For instance, Cannon et a l . (148) found that tRNA bound to 70S ribosomes was r e s i s t a n t to pancreatic RNase. Pestka (162) found that i n the presence of ribosomes and polyU, Phe-tRNA was s u b s t a n t i a l l y protected from h y d r o l y s i s . Although binding of t h i s tRNA to 30S subunits i n response to polyU was subs tan-134 t i a l , the tRNA was not protected from pancreatic RNase diges-t i o n . The presence of both subunits was required f o r sub s t a n t i a l protection of the tRNA from RNase d i g e s t i o n . The data also indicated that the Phe-tRNA which was bound to the ribosomes and r e s i s t a n t to RNase remained i n t a c t , that i s , the aminoacyl end of the aminoacyl-tRNA was protected by the r i b o -some (149 , 255). Neu and Heppel (301) found that ribosomal RNase acted without appreciable destruction of endogenous ribo-somal RNA. G i l b e r t (263) found that i n the presence of polyU, treatment of ribosomes with pancreatic RNase i n the cold had no e f f e c t . Delihas (30 2) found that with his ribosome pre-paration only 3% of the t o t a l absorbance at 260 nm was released i n the presence of pancreatic RNase and there was no e f f e c t on ribosomal a c t i v i t y . Ehresmann and Ebel (303) found that T i RNase caused less degradation i n whole 70S ribosomes than when the treatment was done on 30S and 50S ribosomal subunits. At leas t 35-40% of the 16S RNA was accessible to nuclease a c t i o n . Gupta et a l . (30 4) found that RNase Ti degraded a l l parts of the fz RNA except that protected by the attached ribosomes. Rich's group (305) obtained s i m i l a r r e s u l t s with pancreatic RNase. Equipped with th i s information an extensive characteriza-t i o n of the low molecular weight RNA bound to E_^  c o l i ribosomes was then c a r r i e d out. The r e s u l t s from Table I I I c l e a r l y show that the tRNA bound to WRib has acceptor a c t i v i t y f o r a l l the amino acids. A s i m i l a r phenomenon was observed recently for rabbit r e t i c u l o c y t e ribosomes (276) . The results of Culp et a l . 1 3 5 (276) d i f f e r e d c o n s i d e r a b l y from those shown i n Table 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 which were d i f -f e r e n t again from t h a t bound t o t h e i r ribosomes. Smith and McNamara (277) have a l s o done acceptor 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 those of Culp et a l . (276). This may be due t o d i f f e r e n t i s o l a t i o n t e c h -niques. The r e s u l t s of S t e a m and Horowitz (280) , u s i n g the t o t a l RNA from Neurospora c r a s s a , compare q u i t e favourably w i t h many of the s p e c i f i c a c t i v i t i e s shown i n Table I I I . I t should be s t r e s s e d t h a t the amino a c i d acceptor s t u d i e s were not c a r r i e d out under optimum c o n d i t i o n s . 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 acids t o the tRNAs (2 78). Some synthetases r e q u i r e the presence of a s u l f h y d r y l group w h i l e others do not (275, 279). Since each tRNA species has probably d i f f e r e n t optimal c o n d i t i o n s f o r maximal amino a c i d charging, the present experiments do not, t h e r e f o r e , lead to q u a n t i t a t i v e r e s u l t s . Under the c o n d i t i o n s used, the 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 obtained. The main p o i n t t h a t should be e s t a b l i s h e d here i s t h a t the ribosomes c o n t a i n species of tRNA which have acceptor a c t i v i t y f o r a l l the amino a c i d s and th a t the amount of acceptance f o r each amino a c i d v a r i e s . This may be due t o the r e l a t i v e amounts o f the s p e c i f i c tRNA bound t o the ribosomes, or due t o c o n d i t i o n s under which the assays were c a r r i e d out, or both. B a r t z e t al_. (291) found t h a t there are f l u c t u a t i o n s i n the amounts of s p e c i f i c tRNAs i n c o l i during c e l l growth. For example, tRNA reached i t s maximum 136 Cvs l e v e l i n the stationary phase of c e l l growth whereas tRNA 2 reached i t s maximum l e v e l during the early log phase and then declined a f t e r that. I t i s also possible that d i f f e r e n t ribo-somal preparations w i l l have d i f f e r e n t amounts of s p e c i f i c tRNAs bound or i t could be that c e r t a i n s p e c i f i c tRNAs are more strongly bound to the ribosomes than others. From the previous discussion, i t seems very unlike l y that the v a r i a t i o n i n the r e l a t i v e amounts of s p e c i f i c tRNAs bound to ribosomes i s due to the removal of a portion of some s p e c i f i c tRNAs by the action of exonucleases. The problem of nuclease contamin-ation; for example, finger nucleases, i n various types of systems has occupied the attention of many in v e s t i g a t o r s . As f a r as t h i s laboratory i s concerned, separations of tRNA, for instance on BD-cellulose columns at room temperature for up to four days have been successfully c a r r i e d out without degrada-t i o n of the tRNA and with f u l l retention of the capacity f o r amino acid acceptance. One should also bear i n mind that i t i s presently impossible to c a l c u l a t e , from the available data, the maximum number of tRNA molecules which could be bound to active ribosomes because one would not know what percentage of the ribosomes' was a c t u a l l y f u n c t i o n a l . This i s another area i n which the studies on tRNA bound to ribosomes w i l l have to be pursued i n the future. The d i s t r i b u t i o n of low molecular weight RNA from c o l i ribosomes (Table IV) p a r a l l e l s exactly the results obtained by Busch's group (19 0) for t h e i r f r a c t i o n a t i o n of both low mole-cular weight nucleolar and ribosomal RNA of Novikoff hepatoma 137 ascites c e l l s . According to Tables II and IV the A 2eo r a t i o of 4S : 5S RNA varie d between 0.58 and 0.90 which amounted to 0.9 to 1.3 molecules of tRNA bound to the ribosomes per molecule of 5S RNA. The percentage of t o t a l RNA bound to ribosomes i n the form of tRNA varied between 1.3 and 1.7% while the percentage of the t o t a l RNA bound to ribosomes i n the form of 5S RNA varied between 1.5 and 2.8%. The l a t t e r values i n both of these cases compare quite favourably to those obtained by other investigators (55, 286). S i m i l a r l y , these values compare quite favourably to the expected amounts of rRNA, 5S RNA and tRNA using molecular weights as the only c r i t e r i o n . I t should be borne i n mind that some 4.5S RNA was present i n the 5S RNA fractio n s (Figures 10, 12 and 12a) while only a s l i g h t amount was present i n the 4S RNA fr a c t i o n s (Figures 10 and 25) . For instance, 2.7% of the t o t a l low molecular weight RNA i n the 4-5S RNA region was 4.5S RNA and this was a l l found i n the 5S RNA f r a c t i o n (Figures 12 and 12a). Thus the amount of tRNA bound to the ribosomes calculated on the basis of the amount of 5S RNA should be s l i g h t l y higher. The 4.5S RNA also represents much less than one molecule per molecule of 5S RNA. The 4.5S RNA i n c o l i has been shown by the Cambridge group to be a unique component and not an a r t e f a c t and work i s w e l l underway i n sequencing this p a r t i c u l a r species (30 8). As yet, the function f o r this component has not been c l a r i f i e d . The mobility of t h i s species with respect to 4S and 5S RNA has been v e r i f i e d by Dixon's group (309) . 138 An attempt was made to characterize further the low mole-cular weight RNA bound to c o l i ribosomes by electrophoresis i n a 10% preparative gel (Figure 17) . The main absorbance peak d i d not correspond to the main peak of acceptance a c t i v i t y . E f f o r t s were made to characterize this main absorbance peak. O r i g i n a l l y i t was thought that GTP would be found i n t h i s peak since i t i s a major requirement i n protein synthesis but paper chromatography revealed only two components—one at the o r i g i n i n d i c a t i v e of a large molecular weight species and one moving beyond the GTP standard. The component at the o r i g i n gave the t y p i c a l RNA tra c i n g (Figure 19). Further attempts to charac-t e r i z e t h i s peak by chromatography on DEAE-cellulose or DEAE-Sephadex A-50 columns resulted i n only a s i n g l e peak being eluted i n the void volume i n each case. At the present moment the chara c t e r i z a t i o n of the main absorbance peak remains to be established. Besides containing some tRNA, this p r i n c i p a l peak must contain RNA of lower mole-cular weight since presumably t h i s would be eluted f i r s t from the preparative g e l . I t i s quite possible that t h i s RNA i s actually tRNA that has been p a r t i a l l y degraded. In order to confirm t h i s i t would be necessary to separate this RHA from the i n t a c t tRNA but unfortunately t h i s cannot be done r e a d i l y . A l k a l i n e hydrolysis studies of t h i s RNA species should confirm whether the sample i s tRNA but unfortunately i n s u f f i c i e n t material was availa b l e to carry out the analyses. Because of the experimental d i f f i c u l t i e s and the l i m i t e d amount of i n f o r -mation that could have been obtained the cha r a c t e r i z a t i o n of 139 t h i s peak was l e f t for future consideration. At t h i s stage i n the thesis the following a d d i t i o n a l points had been established i n addition to those previously mentioned (see p. 132-133): (a) the ribosomes contain species of tRNA which have acceptor a c t i v i t y for a l l the amino acids and that the amount of acceptance for each amino acid varied, (b) 1-2 molecules of tRNA are bound to the ribosomes per mole-cule of 5S RNA, (c) the presence of 4.5S RNA bound to c o l i WRib was confirmed and this species represented much less than one molecule per molecule of 5S RNA. According to my hypothesis on chain-termination, which was thoroughly discussed previously, i f a terminating tRNA exists i t must be t i g h t l y bound to the ribosomes and probably i t would occupy a s i t e on the ribosome d i s t i n c t from the nor-mal tRNA s i t e s . Such a species of RNA would not be expected to exchange with the normal tRNA. Therefore, exchange experi-ments were c a r r i e d out with two purposes i n mind: (1) to see i f some tRNAs are more t i g h t l y bound to ribosomes than others, and (2) to confirm the presence or absence of a terminating tRNA. I t had been shown by numerous investigators that exchange between free tRNA and tRNA bound to ribosomes rea d i l y takes place (148, 249, 250, 306). This exchange was not affected by charging tRNA with amino acids or by temperature. Bound tRNA w i l l not wash of f i n high Mg + + ion concentration but could e a s i l y be displaced by free tRNA from the surrounding medium. The nonspecific association of tRNA and ribosomes took place i n 140 the cold and d i d not require the supernatant enzymes, GTP, ATP or an energy source other than the thermal energy of the react-ing components. An exchange experiment was c a r r i e d out i n the same manner as by Cannon et a l . (148). Ribosomes were prepared from a pyrimidine-requiring c o l i mutant grown i n the presence of 3 H - u r a c i l and thoroughly washed i n the usual manner. Exchange was considered to be complete when the number of counts i n the ribosomal p e l l e t had remained v i r t u a l l y constant w i t h i n experi-mental error (Table V, Figure 23). When l a b e l l e d ribosomal-bound tRNA was exchanged with unlabelled tRNA v i r t u a l l y a l l of the l a b e l l e d tRNA was removed (Table V I ) . The A 2 6 o r a t i o of 4S : 5S RNA on a t o t a l ribosomal RNA basis had increased to almost 3.5 i n ribosomes exchanged with unlabelled tRNA (Figure 24). This represented about 5.7 molecules of tRNA bound per molecule of 5S RNA as compared with 1-2 molecules of tRNA bound i n a system that has not undergone exchange. These results may be explained by considering a m u l t i p l i c i t y of tRNA binding s i t e s previously postulated by Warner and Rich (260) as w e l l as by Wettstein and N o l l (262) for mammalian ribosomes (see Introduction p. 22-24) . Quantitative studies by Cannon et a l . (14 8) showed that there was only one binding s i t e per 70S or 50S ribosome i n the absence of mRNA. In the presence of messenger the amount of binding doubled. Other investigators (285) have suggested the presence of three s i t e s , two f o r amino-acyl-tRNA and one f o r peptidy1-tRNA. The second aminoacyl-tRNA s i t e was occupied only i n the absence of peptide bond formation. 141 A c c o r d i n g t o C a n n o n e t a l _ . (148) t h i s d o e s n o t e l i m i n a t e t h e 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 t h e y a r e n o t a l l e q u i v a l e n t , o r i f t h e y a r e e q u i v a l e n t t h e y h a v e t h e p r o p e r t y t h a t b i n d i n g t R N A t o a n y o n e w e a k e n s t h e b i n d i n g a b i l i t y o f t h e o t h e r s . T h i s m i g h t a c c o u n t f o r t h e l o s s o f l a b e l l e d r i b o s o m a l b o u n d t R N A . Two m o d e l s a r e s u g g e s t e d f o r t h e b i n d i n g a n d e x c h a n g e o f t R N A . I n t h e f i r s t , t h e r i b o s o m e h a s o n e s i t e o n w h i c h t h e t R N A b i n d s i n r a p i d e q u i l i b r i u m w i t h t h e t R N A i n s o l u t i o n . T h e s e c o n d m o d e l c a n b e c h a r a c t e r i z e d a s t h e r i b o s o m e h a v i n g a n e x c h a n g e s i t e . T h e t R N A b o u n d t o t h e s i t e i s t i g h t l y b o u n d a n d t h e c o m p l e x w o u l d d i s s o c i a t e v e r y s l o w l y . T h e e x c h a n g e w o u l d t a k e p l a c e b y a s e c o n d t R N A m o l e c u l e a l t e r i n g t h e s i t e a n d d i s p l a c i n g t h e f i r s t . O n e c a n c o n s i d e r t w o e q u i v a l e n t s i t e s f o r t h e b i n d i n g ; e i t h e r s i t e a l o n e c a n b i n d t h e t R N A t i g h t l y b u t i f b o t h a r e f u l l b o t h t R N A m o l e c u l e s a r e b o u n d l o o s e l y . S u c h a s t r u c t u r e w o u l d h a v e r a p i d e x c h a n g e a s s o c i a t e d w i t h a l o o s e b i n d i n g f o r t h e t w o m o l e c u l e s b u t s l o w l o s s a n d t i g h t b i n d i n g f o r t h e f i r s t t R N A b o u n d . A c o m p l e t e d i s c u s s i o n o f t h e n u m b e r o f s i t e s a v a i l a b l e o n t h e r i b o s o m e f o r b i n d i n g t R N A h a s a l r e a d y b e e n g i v e n ( p a g e s 14-24). T h e d a t a p r e s e n t e d h e r e d o n o t e x c l u d e t h e p o s s i b i l i t y t h a t o t h e r k i n d s o f t R N A b i n d i n g s i t e s w i t h d i f f e r e n t r e q u i r e -m e n t s c o u l d b e d e m o n s t r a t e d u n d e r d i f f e r e n t c o n d i t i o n s . U n f o r t u n a t e l y a f t e r t h e l a s t e x c h a n g e ( F i g u r e 23) t h e r i b o s o m e s w e r e f o u n d t o b e i n a c t i v e i n p r o t e i n s y n t h e s i s a l t h o u g h a f t e r t h e i n i t i a l e x c h a n g e t h e s e r i b o s o m e s w e r e a s a c t i v e a s t h e o r i g i n a l p r e p a r a t i o n . Many r e a s o n s c o u l d e x p l a i n 142 t h i s loss i n a c t i v i t y . For example, because of the increased number of tRNAs bound to the ribosomes as compared to 5S RNA, a l l the a v a i l a b l e s i t e s on the ribosome may be blocked and/or the conformation of the ribosome may be altered i n such a way as to prevent the normal exchange from taking place and there-fore preventing protein synthesis. I t has also been found that 5S RNA could be released from the 50S ribosomal subunit by high l e v e l s of tRNA (287). Sarkar and Comb (288) could give no explanation for this phenomenon. Their only suggestion was that i n c e l l s where most of the ribosomes were involved i n protein synthesis, 5S RNA may have occupied one of two s i t e s and at one of these s i t e s i t could be displaced by tRNA. Their previous studies on 80S ribosomes demonstrated that two mole-cules of 5S RNA were bound per large subunit but only one molecule per subunit when tRNA was included i n the binding mix-ture (2 88) . They also found that with the loss of 5S RNA there was a loss i n b i o l o g i c a l a c t i v i t y . They suggest that the loss i n b i o l o g i c a l a c t i v i t y may have been due to s l i g h t changes i n the conformation of the ribosome and may have no r e l a t i o n s h i p to the loss of 5S RNA. The f a c t that native 5S RNA present on the 50S ribosomal subunits, when exchanged with i s o l a t e d 5S RNA, 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 polypeptide synthesis, suggested to them that the conformation of the 5S RNA on the ribosome was unique and e s s e n t i a l for b i o l o g i c a l a c t i v i t y and quite d i f f e r e n t from i s o l a t e d 5S RNA. Other possible reasons for the loss i n b i o l o g i c a l a c t i v i t y could be due to a conformational change i n a p r o t e i n , a l o c a l rearrange-143 merit of a h e l i c a l p o r t i o n of the RNA or 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 ribosomal RNase i s remote due to the f a c t t h a t the exchange took place at a high magnesium c o n c e n t r a t i o n and at 0°; c o n d i t i o n s underwhich the ribosomes are q u i t e s t a b l e . Although a f t e r the exchange experiment the ribosomes were found t o be i n a c t i v e and i d e a l l y one would have hoped f o r the maintenance of a c t i v i t y , the u l t i m a t e purpose of the experiment was t o study the 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 these ribosomes and t o check f o r the presence of a t e r m i n a t i n g tRNA— both of which could s t i l l be s t u d i e d (see p. 139). Therefore a f t e r the exchange, the ribosomal-bound 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 see i f the l a b e l l e d tRNA t h a t remained bound t o c o l i ribosomes was spread evenly throughout the e l u t i o n p r o f i l e , or whether i t was concentrated i n a c e r t a i n r e g i o n . The l a b e l l e d tRNA was spread uniformly throughout the reg i o n e l u t e d w i t h the NaCl g r a d i e n t but there was a peak of r a d i o a c t i v i t y i n the eth a n o l g r a d i e n t (Figure 26). Counts i n the 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 . This peak ethanol 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 thanol f r a c t i o n were much hig h e r than those recorded i n the s a l t g r a d i e n t most of i t could be accounted f o r i n the f o l l o w i n g way: (1) the presence of 4.5S RNA which would be l a b e l l e d . This 4.5S RNA, which would tend t o remain s t r o n g l y bound to the B D - c e l l u l o s e column, would be e l u t e d i n high ethanol concentrations which would overcome the hydrophobic 144 i n t e r a c t i o n s between the RNA and the r e s i n , (2) since the ethanol f r a c t i o n has been found to contain a l l Ser, Tyr and Trp acceptor a c t i v i t i e s , about 17% of the Leu acceptor a c t i v i t y and 9% of the Phe acceptor a c t i v i t y (295) , this region alone would be expected to give higher counts due to t h e i r combined presence i n t h i s f r a c t i o n (see Table I I I ) , (3) since tRNA T r p was found to be present i n the lar g e s t amount on ribosomes, presumably i t alone could account for some of the counts (Table I I I ) ; however, there i s no reason to b e l i e v e that one p a r t i c u l a r tRNA i s less vulnerable to exchange than another. The reasons a d d i t i o n a l experiments to confirm these r e s u l t s could not be c a r r i e d out were: (a) acceptor studies with Trts tRNA - gives a very high background (up to 300 cpm) and since one i s dealing with such low counts (250 cpm), i t would be impossible to get a d e f i n i t i v e r e s u l t , (b) d o u b l e - l a b e l l i n g experiments could not be c a r r i e d out because the background levels would be higher than the counts that were being i n v e s t i -gated here, (c) the assay used to determine acceptance a c t i v i t y of s p e c i f i c tRNAs would involve the p r e c i p i t a t i o n of a l l the tRNAs i n the ethanol f r a c t i o n . I t may be possible to separate the 5 acceptor a c t i v i t i e s found i n the ethanol f r a c t i o n on a Kelmer reversed-phase column but, once again, since one i s dealing with such low counts any small error, f o r example, i n background counts, would lead to an o v e r a l l large experimental e r r o r . These experiments would have been very complex and time consuming and i t was f e l t that any results obtained would not have added 145 s i g n i f i c a n t l y to the conclusions already established. Taking a l l these factors i n t o consideration, there would be very few counts l e f t over which could be al l o c a t e d to the presence of a s p e c i f i c chain-terminating tRNA. This species of tRNA would not only be l a b e l l e d but would also be expected to be present as a sharp peak of r a d i o a c t i v i t y . Approximately 50,000 counts a t t r i b u t e d mainly to tRNA was d i s t r i b u t e d over the BD-cellulose column (Figure 24, Table VI). I f a mere 1% of t h i s tRNA could be considered due to the presence of a s p e c i f i c tRNA terminator, then there would be enough radio-a c t i v i t y present such that i t would show up as a d i s t i n c t sharp peak. Ca l c u l a t i n g on the basis of 5S RNA, the counts remaining would in d i c a t e less than one molecule of 4S material for every 20 5S RNA molecules. Thus the presence of a s p e c i f i c t i g h t l y bound terminating tRNA i s not possible. i In summary, tRNA exchange resulted i n increased binding of tRNA to the ribosomes; from 1-2 molecules to approximately 6 molecules per molecule of 5S RNA. Exchange, under the conditions used, was almost 100% and also resulted i n the loss of ribosomal a c t i v i t y . The tRNA which d i d not exchange was spread uniformly throughout the e l u t i o n pattern suggesting that a l l tRNAs are probably bound to the ribosome to the same degree, that i s , one tRNA i s not bound more strongly than an-other. The absence of a sharp peak of r a d i o a c t i v i t y i s further proof of the absence of a s p e c i f i c chain-terminating tRNA, since as previously discussed, such a species of tRNA would be expected to be not only t i g h t l y bound to the ribo-somes but also non-exchangeable with tRNA. 146 BIBLIOGRAPHY 1. Zamecnik, P . C , C o l d S p r i n g Harbor Sym. Quant. B i o l . , 34, 1 (1969). 2. T i s s i e r e s , A., and Watson, J.D., Nature, 182, 778 (1958). 3. B o l t o n , E.T., Hoyer, W.H., and R i t t e r , D.B., i n R.B. Roberts ( E d i t o r ) , Microsomal 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 York, 1958, p. 18. 4. Roberts, R.B. ( E d i t o r ) , i b i d . 5. Roberts, R.B., B r i t t e n , R.J., and B o l t o n , E.T., i b i d . , p. 84. 6. T i s s i e r e s , A., Watson, J.D., S c h l e s s i n g e r , D., and H o l l i n g w o r t h , B.R., J . Mol. B i o l . , 1, 221 (1959). 7. K u r l a n d , C.G., J . Mol. B i o l . , 2, 83 (1960). 8. G o r i n i , L., and K a t a j a , E., Proc. Nat. Acad. S c i . U.S.A., 51, 487 (1964). 9. G u t h r i e , C., and Nomura, M., Nature, 219, 232 (1968). 10. H i l l e , M.B., M i l l e r , M.J., Iwasaki, K., and Wahba, A . J . , Proc. Nat. Acad. S c i . U.S.A., 5_8, 1652 (1967). 11. Nomura, M., and Lowry, C V . , Proc. Nat. Acad. S c i . U.S.A., 58, 946 (1967). -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., and Gros, F., Biochem. Biophys. Res. Commun., 25, 124 (1966). 14. S t a n l e y , W.M., J r . , S a l a s , M., Wahba, A . J . , and Ochoa, S., Proc. Nat. Acad. S c i . U.S.A., 5_6 , 290 (1966). 15. R e v e l , M. , L e l o n g , J . C , Brawerman, G. , and Gros, F. , Nature, 219_, 1016 (1968). 16. Wahba, A . J . , Chae, Y.B., Iwasaki, K., Mazumder, R., M i l l e r , M.J., S a b o l , S., and S i l l e r o , M.A.G., C o l d S p r i n g Harbor Sym. Quant. B i o l . , 3_4, 285 (1969). 17. C l a r k , B . F . C , and Marcker, K.A., J . Mol. B i o l . , 17, 394 (1966) . 18. S t e i t z , J.A., Nature, 224, 957 (1969). 147 19. H i n d l e y , J . , and S t a p l e s , D.H., Nature, 2_2_4, 964 (1969). 20. Herzberg, M. , L e l o n g , J . C , and R e v e l , M. , J . Mol. B i o l . , _4_4, 297 (1969). 21. Greenshpan, H., and R e v e l , M., Nature, 224, 331 (1969). 22. Lucas-Lenard, J . , and Lipmann, F., Proc. Nat. Acad. S c i . U.S.A., 57, 1050 (1967). 23. Ghosh, H.P., and Khorana, H.G., Proc. Nat. Acad. S c i . U.S.A., 58, 2455 (1967). 24. Nomura, M. , Lowry, C.V., and G u t h r i e , C , Proc. Nat. Acad. S c i . U.S.A., 5_8, 1487 (1967). 25. K o l a k o f s k y , D., Ohta, T., and Thach, R.E., Nature, 220, 244 (1968). 26. E r t e l , R., B r o t , N., R e d f i e l d , B., A l l e n d e , J . E . , and Weissbach, H. , Proc. Nat. Acad. S c i . U.S.A., 5_9, 861 (1968). 27. Ono, Y., S k o u l t c h i , A., K l e i n , A., and L e n g y e l , P., Nature, 220, 1304 (1968). 28. Ono, Y., S k o u l t c h i , A., Waterson, J . , and L e n g y e l , P., Nature, 222, 645 (1969). 29. Grunberg-Manago, M., C l a r k , B.F.C., R e v e l , M., Rudland, P.S., and Dondon, J . , J . Mol. B i o l . , 40_, 33 (1969). 30. Monro, R.E., J . Mol. B i o l . , 26_, 147 (1967). 31. Lucas-Lenard, J . , and Haenni, A., Proc. Nat. Acad. S c i . U.S.A. , 6_3, 93 (1969) . 32. Erbe, R.H., Nau, M., and Leder, P., J . Mol. B i o l . , 39, 441 (1969). 33. Craven, G.R., Voynow, P., Hardy, S.J.S., and K u r l a n d , C. G. , B i o c h e m i s t r y , 8, 2906 (1969). 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. , Pearson, P., and T i s s i e r e s , A., C o l d S p r i n g Harbor Sym. Quant. B i o l . , 34, 25 (1969). 35. Moore, P.D., T r a u t , R.R., N o l l e r , H., Pearson, P., and D e l i u s , H., J . Mol. B i o l . , 31, 441 (1968). 36. K u r l a n d , C.G., Voynow, P., Hardy, 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 Harbor Sym. Quant. B i o l . , 34, 17 (1969). 148 37. Sypherd, P.S., O ' N e i l l , D.M., and Taylor, M.M., Cold Spring Harbor Sym. Quant. B i o l . , 3_4, 77 (1969) . 38. Mizushima, S., arid Nomura, M., Nature, 226, 1214 (1970). 39. Hershey, J.W.B., Dewey, K.F., and Thach, R.E., Nature, 222, 944 (1969). 40. Parenti-Rosina, R., Eisenstadt, A., and Eisenstadt, 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 , B.G., Nature, 215, 735 (1967). 43. K l e i n , H.A., and Capecchi, M.R., J . B i o l . Chem., 246, 1055 (1971). 44. Stanley, W.M., J r . , and Bock, R.M., Biochemistry, £, 1302 (1965). 45. Midgley, J.E.M., Biochem. Biophys. Acta, 10 8, 348 (1965). 46. Mcllreavy, D.J., and Midgley, J.E.M., Biochem. Biophys. Acta, 142, 47 (1967). 47. F e l l n e r , P., and Sanger, F., Nature, 219, 236 (1968). 48. Smith, I., Dubnau, D., More l l , P., and Marmur, J . , J . Mol. B i o l . , 33, 123 (1968). 49. Aronson, A.I., J . Mol. B i o l . , 5, 453 (1962). 50. Takanami, M., J . Mol. B i o l . , 23, 135 (1967). 51. Moller, W., and Boedtker, H., Editions du Centre National 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. Leppla, S.H., Ph.D. Thesis, University of Wisconsin, Madison, Wisconsin (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 . Soc. Chim. B i o l . , 46, 87 (1964). 56. Zehavi-Willner, T., and Comb, D.G., J . Mol. B i o l . , 16, 250 (196.6). 149 57. Young, R.J., B i o c h e m i s t r y , 7, 2263 ( 1 9 6 8 ) . 58. F e l l n e r , P., Ehresmann, C., and E b e l , J.P., Nature, 2 2 5 , 26 ( 1 9 7 0 ) . 59. Doty, P., Boedtker, H., F r e s c o , J.R., Haselkorn, R., and L i t t , M. , Proc. Nat. Acad. S c i . U.S.A., 45^, 482 ( 1 9 5 9 ) . 60. Traub, P., and Nomura, M. , J . Mol. B i o l . , 3_4, 575 ( 1 9 6 8 ) . 6 1 . Traub, P., and Nomura, M. , J . Mol. B i o l . , 40_, 391 ( 1 9 6 9 ) . 62. Furano, A.V., B r a d l e y , D.F., and 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. Santer, M. , and Smith, J.R. , J . B a c t e r i o l o g y , 9_2, 1099 ( 1 9 6 6 ) . 64. Geiduschek, P., and Has e l k o r n , R., Ann. Rev. Biochem., _38, 647 (1969) . 65. A t t a r d i , G., Ann. Rev. M i c r o b i o l o g y , 21, 383 ( 1 9 6 7 ) . 66. M a t t h a e i , H., Sander, G., Swan, O., Kreuzer, T., C a f f i e r , H. , and Parmeggiani, A., N a t u r w i s s e n s c h a f t e n , 5_5, 281 ( 1 9 6 8 ) . 6 7 . Ochoa, S., Naturwissenschaf t e n , 5_5, 505 ( 1 9 6 8 ) . 68. C o l d S p r i n g Harbor Sym. Quant. B i o l . , 3 1 , ( 1 9 6 6 ) . 69. Ron, E.Z., K o h l e r , R.E., and D a v i s , B.D., J . Mol. B i o l . , 36, 83 ( 1 9 6 8 ) . 70. S p i r i n , A.S., and G a v r i l o v a , L.P., i n The Ribosome, S p r i n g e r - V e r l a g New York, Inc., New York, 19 69. 71. Marcker, K.A. , and Sanger, F. , J . Mol. B i o l . , 8, 835 ( 1 9 6 4 ) . 72. C l a r k , B.F.C., and Marcker, K.A., Nature, 2 1 1 , 378 ( 1 9 6 6 ) . 73. Adams, J.M., and Ca p e c c h i , M., Proc. Nat. Acad. S c i . U.S.A. , 5_5, 147 (1966) . 74. Webster, R., E n g e l h a r d t , D., and Z i n d e r , N., Proc. Nat. Acad. S c i . U.S.A., 5_5, 155 ( 1 9 6 6 ) . 75. C a p e c c h i , M. , Proc. Nat. Acad. S c i . U.S.A., 5_5, 1517 (1966) . 76. Smith, A.E., and Marcker, K.A., J . Mol. B i o l . , 38, 241 ( 1 9 6 8 ) . 150 77. Galper, J.B., and D a r n e l l , J.E., Biochem. Biophys. Res. Commun., 34, 205 (1968). 78. Schwartz, J.H., Meyer, R., E i s e n s t a d t , J.M., and Brawerman, G., J . Mol. B i o l . , 25, 571 (1967). 79. Bachmayer, H., and K r e i l , G., Biochim. Biophys. A c t a , 169, 95 (1968). 80. Bruton, C.J., and H a r t l e y , B.S., Biochem. J . , 108, 281 (1968). 81. Dickerman, H.W., S t e e r s , E., J r . , R e d f i e l d , B.G., and Weissbach, 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., Proc. Nat. Acad. S c i . U.S.A., 61, 1066 (1968). 83. N i s h i z u k a , Y., and Lipmann, F., Arch. Biochem. Biophys., 116, 344 (1966). 84. B r e t s c h e r , M.S., J . Mol. B i o l . , 3£, 131 (1968). 85. Zinder, N.D., Engelhardt, D.L., and Webster, R.E., Cold Spring Harbor Sym. Quant. B i o l . , 3_1, 251 (1966) . 86. Fox, J.L., and Ganoza, M.C., Biochem. Biophys. Res. Commun., 32, 1064 (1968). 87. S o l i , D., J . Mol. B i o l . , 34, 175 (1968). 88. Caskey, C.T., Tompkins, R., S c o l n i c k , E., Caryk, T., and Nirenberg, M., Science, 162, 135 (1968). 89. S c o l n i c k , E., Tompkins, R., Caskey, T., and Nirenberg, M., Proc. Nat. Acad. S c i . U.S.A., 61, 765 (1968). 90. Milman, G., G o l d s t e i n , J . , S c o l n i c k , E., and Caskey, T., Proc. Nat. 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., Proc. Nat. Acad. S c i . U.S.A., 6±, 1235 (1969). 92. G o l d s t e i n , J . , Milman, G., S c o l n i c k , E., and Caskey, T., Proc. Nat. 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., Proc. Nat. Acad. S c i . U.S.A., £5, 702 (1970). 94. Caskey, T., S c o l n i c k , E., Tompkins, R. , G o l d s t e i n , J . , and Milman, G., Cold Spring Harbor Sym. Quant. B i o l . , 34, 479 (1969). 151 95. Smrt, J . , Kemper, W., Caskey, T., and Nirenberg, M., J. B i o l . Chem., 245, 2753 (1970). 96. Beaudet, A.L., and Caskey, C T . , Nature, 227, 38 (1970). 97. G o l d s t e i n , J.L., Beaudet, A.L., and Caskey, C T . , Proc. 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 . , Proc. Nat. Acad. S c i . U.S.A., 67, 537 (1970). 99. S c o l n i c k , E., Milman, G., Rosman, M., and Caskey, T., Nature, 225, 152 (1970). 100. Ganoza, M.C, and Tomkins, J.K.N., Biochem. Biophys. Res. Commun., 40, 1455 (1970). 101. Vo g e l , Z., Zamir, 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., Greenberg, R.E., Hurwitz, R., and C h a p e v i l l e , F., Proc. Nat. Acad. S c i . U.S.A., 58, 2079 (1967). 103. deGroot, N., Panet, A., and L a p i d o t , Y., Biochem. Biophys. Res. Commun., 31, 37 (1968). 104. Vo g e l , Z., Zamir, A., and E l s o n , D., Proc. Nat. Acad. S c i . U.S.A., 61, 701 (1968). 105. K o s s e l , M., and Raj Bhandary, U.L., J . Mol. B i o l . , 35, 539 (1968). 106. Menninger, J.R., Mu l h o l l a n d , M.C, and S t i r e w a l t , W.S., Biochem. Biophys. A c t a , 217, 496 (1970). 107. Capecchi, M.R., and K l e i n , H.A., Nature, 226, 1029 (1970). 108. N i c h o l s , J.L., Nature, 225, 147 (1970). 109. I s h i t s u k a , H., and K a j i , A., Proc. Nat. Acad. S c i . U.S.A., 66, 168 (1970). 110. Subramanian, A.R., Ron, E.Z., and Davis, B.D., Proc. Nat. Acad. S c i . U.S.A., 61, 761 (1968). 111. Subramanian, A.R., and Davis, B.D., Nature, 228, 1273 (1970). 112. Sabol, S., S i l l e r o , M.A.G., Iwasaki, K., and Ochoa, S., Nature, 228, 1269 (1970). 152 113. Subramanian, A.R., D a v i s , B.D., and B e l l e r , R.J., Cold Spr 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., Gonzalez, N.S., and Bade, E.G., Proc. 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., Proc. Nat. Acad. S c i . U.S.A., 58, 1782 (1967). 116. Friedman, H., Lu, P., and R i c h , A., Nature, 223, 909 (1969). 117. Iwasaki, K., Sabol, S., Wahba, A.J., and Ochoa, S., Arch. Biochem. Biophys., 125, 542 (1968). 118. Chae, Y.B., Mazumder, R., and Ochoa, S., Proc. 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., Proc. Nat. Acad. S c i . U.S.A., 67, 1137 (1970). 120. Kaempfer, R., Nature, 228, 534 (1970). 121. Takata, R., Osawa, S., Tanaka, K., Teraoka, H., and Tamaki, M., Molec. Gen. Genetics, 109, 123 (1970). 122. Weisblum, B., and Davies, J . , B a c t e r i o l . Reviews, 32, 493 (1968). 123. Kaltschmidt, E., and Wittman, H.G., Proc. Nat. Acad. S c i . U.S.A., 6_7, 1276 (1970). 124. Osawa, S., Takata, R., and Dekio, S., Molec. Gen. Genetics, 107, 32 (1970) . 125. Ozaki, M., Mizukshima, S., and Nomura, M., Nature, 221, 333 (1969). 126. Slobin, L . I . , Biochem. Biophys. Res. Commun., 3_9_, 470 (1970). 127. Stanley, W.M. , J r . , and Bock, R.M. , Biochemistry, 4_, 1302 (1965). 128. K i r t i k a r , D.M.W., and K a j i , A., J . B i o l . Chem., 243, 5345 (1968) . 129. Forget, B.G., and Weissman, S.M., Science, 158, 1695 (1967). 130. Jordan, B.R., Feunteun, J . , and Monier, R., J . Mol. B i o l . , 5£, 605 (1970). 153 131. S i d d i q u i , M.A.Q., and Hosokawa, K., Biochem. Biophys. Res. Commun., 36, 711 (1969). 132. Raacke, I.D., Biochem. Biophys. Res. Commun., 31, 528 (1968). 133. Cantor, C.R., Nature, 216, 513 (1967). 134. Boedtker, H., and K e l l i n g , D.G., Biochem. Biophys. Res. Commun., 29-, 758 (1967). V 135. Lewis, J.B., and Doty, P., Nature, 225, 510 (1970). 136. W i l l i a m s o n , R., and Brownlee, G.G., Fed. Eur. 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 . Mol. B i o l . , 5_3, 357 (1970). 138. M i l l e r , O.L., Hankalo, B.A., and Thomas, C.A., J r . , Science, 169, 392 (1970). 139. Purdom, I . , Bishop, J.O., and B u n s t i e l , M.L., Nature, 227, 239 (1970). 140. Muto, M., Biochemistry, 9, 3683 (1970). 141. J a r r y , B., and Rosset, R., Biochem. Biophys. Res. Commun., 41, 789 (1970). 142. Ehresmann, C., F e l l n e r , P., and E b e l , J.B., Nature, 227. 1321 (1970). 143. Hartman, K.A., and Thomas, G.J., J r . , Science, 170, 740 (1970). 144. Nomura, M., and Erdmann, V.A., Nature, 228, 744 (1970). 145. G u t h r i e , C., Nashimoto, H., and Nomura, M., Cold S p r i n g Harbor Sym. Quant. B i o l . , 3_4, 69 (1969). 146. Nashimoto, H., and Nomura, H., Proc. Nat. Acad. S c i . U.S.A., 67, 1440 (1970). 147. Takanami, M., J . Mol. B i o l . , 23 135 (1967). 148. Cannon, M. , Krug, R. , and G i l b e r t , W. , J . Mol. B i o l . , 1_, 360 (1963). 149. K a j i , A., and K a j i , H., Biochem. Biophys. Res. Commun., 13, 186 (1963). 154 150. K a j i , H., and K a j i , A., Proc. Nat. Acad. S c i . U.S.A., . 52, 1541 (1964). 151. S p y r i d e s , G.J., Proc. Nat. Acad. S c i . U.S.A., 51, 1220 (1964). 152. P e s t k a , S. , and Nirenberg, M.W. , J . Mol. B i o l . ,''21, 145 (1966). 153. Kurland, C.G., J . Mol. 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 Benzer, S., Proc. Nat. Acad. S c i . U.S.A., £8, 1086 (1962). 156. Suzuka, I . , K a j i , H., and K a j i , A., Proc. Nat. 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. B i o p h y s R e s . Commun., 29_, 593 (1967). 158. Vazquez, D., and Monro, D.E., Biochim. Biophys. A c t a , 142, 155 (1967). 159. P e s t k a , S., and Nirenberg, M. , J . Mol. B i o l . , 21, 145 (1966). 160. Monro, R.E., J . Mol. B i o l . , 26, 147 (1967). 161. P e s t k a , S., J . B i o l . Chem., 242, 4939 (1967). 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. McLaughlin, C.S., Dondon, J . , Grunberg-Manago, M., Mic h e l s o n , A.M., and Saunders, G., J . Mol. B i o l . , 32, 521 (1968). 165. Monro, R.E., Cerna, J . , and Marcker, K.A., Proc. Nat. Acad. S c i . U.S.A., 61, 1042 (1968). 166. K a j i , H., Suzuka, I . , and K a j i , A., J . Mol. B i o l . , 18, 219 (1966). 167. Moore, P.B., J . Mol. B i o l . , 18, 8 (1966). 168. Furano, A.V., Biochim. Biophys. A c t a , 161, 255 (1968). 155 169. Traut, R.R. , and Haenni, A.L. , Europ. J . Biochem.. 2_, 64 (1967). 170. Knight, E., J r . , and Sugiyama, T., Proc. Nat. Acad. S c i . U.S.A. , 6_3, 1383 (1969) . 171. Knight, E. , J r . , Biochemistry, 8_, 5089 (1969). 172. Nass, M.M.K., and Buck, C A . , Proc. Nat. Acad. S c i . U.S.A., 62, 506 (1969). 173. Barnett, W.E., and Brown, D.H., Proc. Nat. Acad. S c i . U.S.A., 57, 452 (1967). 174. Nass, M.M.K., and Buck, C A . , J . Mol. B i o l . , 5_4, 187 (1970). 175. Pere, J . J . , Knight, E., J r . , and Darnell, J.E., J . Mol. 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 (1969). 177. Forget, B.G., and Weissman, S.M., Nature, 213, 878 (1967) 178. Sirbasku, D.A., and Buchanan, J.M., J . B i o l . Chem., 245, 2693 (1970). 179. Bishop, J.M., Levinson, W.E., Q u i n t r e l l , N., S u l l i v a n , D. , Fanshier, L. , and Jackson, J . , Virology, 42!, 182 (1970). 180. Bishop, J.M., Levinson, W.E., Su l l i v a n , D., Fanshier, L., Q u i n t r e l l , W. , and Jackson, J . , Virology, 42_, 927 (1970). 181. Weinberg, R.A. , and Penman, S., J . Mol. B i o l . , 3_8, 289 (1968). 182. Weinberg, R.A., and Penman, S., Biochim. Biophys. Acta, 190, 10 (1969). 183. Bernhardt, D., and Darnell, A.E., J r . , J . Mol. B i o l . , 42, 43 (1969). 184. Mowshowitz, D.B. , J . Mol. B i o l . , 50_, 143 (1970). 185. Gardner, J.A.A., and Hoagland, M.B., J . Mol. B i o l . , 243, 10 (1968). 186. King, H.W.S., and Fitschen, W., Biochim. Biophys. Acta, 155, 32 (1968). 156 187. Zapisek, W.F., Saponara, A.G., and Enger, M.D., Biochem-i s t r y , 8, 1170 (1968) . 188. Muramatsu, M., Hodnett, J.L., and Busch, H., J . B i o l . Chem., 241, 1544 (1966). 189. Nakamura, T., Rapp, F., and Busch, H., Cancer Res., 27, 1084 (1967). 190. Nakamura, T., P r e s t a y k o , A.W., and Busch, H., J . B i o l . Chem., 243, 1368 (1968). 191. Peacock, A . C , and Dingman, CW. , B i o c h e m i s t r y , 6_, 1818 (1967) . 192. Hodnett, J.L., and Busch, H., J . B i o l . Chem., 243, 6334 (1968) . 193. P r e s t a y k o , A.W., Tonato, M., and Busch, H., J . Mol. B i o l . , 47, 505 (1970). 19 4. Ro-Choi, T.S., Moriyama, Y., C h o i , Y . C , and Busch, M., J . B i o l . Chem., 245, 1970 (1970). 195. Moriyama, Y., l p , P., and Busch, H., Biochim. Biophys. A c t a , 209, 161 (1970). 196. E l - K h a t i b , S.M. , Ro-Choi, T.S., C h o i , Y . C , and Busch, H., J . B i o l . Chem., 245, 3416 (1970). 197. Busch, H., i n H. Busch and K. Smetana ( E d i t o r s ) , The N u c l e o l u s , Academic P r e s s , New York, 19 70, p. 285. 198. P r e s t a y k o , A.W., Tonato, M., Lewis, B.C., and Busch, H., J . B i o l . Chem., 246, 182 (1971). 199. Sy, J . and McCarty, K.S., Biochim. Biophys. A c t a , 228, 517 (1971). 200. Udem, S.A., Kaufman, K., and Warner, J.R., J . B a c t e r i o l o g y , 105, 101 (1971) . 201. L i z a r d i , P.M., and Luck, D.J.L., Nature New B i o l o g y , 229, 140 (1971). 202. Brownlee, G.G., Nature New B i o l o g y , 229, 147 (1971). 203. H i n d l e y , J . , J . Mol. B i o l . , 30_, 125 (1967). 204. G o l d s t e i n , J . , and Harewood, K. , J . Mol. B i o l . , 3_9, 383 (1969). 157 205. Altman, S., Nature New B i o l o g y , 229, 19 (1971). 206. Iwabuchi, M., Mizukami, Y., and Sameshima, M., Biochim. Biophys. A c t a , 228, 693 (1971). 207. Laycock, D.G., and Hunt, J.A., Nature, 221, 1118 (1969). 208. L a b r i e , F., Nature, 221, 1217 (1969). 209. Dubuy, B., and Weissman, S.M., J . B i o l . Chem., 246, 747 (1971). 210. Voynow, P., and Kurland, C.G., B i o c h e m i s t r y , 10_, 517 (1971). 211. Scraup, H.W., Green, M., and Kurland, C.G., Mol. Gen. G e n e t i c s , 109, 193 (1970). 212. L o d i s h , H.F., Nature, 226, 705 (1970). 213. Adams, J.M., Jeppesen, P.G.N., Sanger, F., and B a r r e l l , B., Nature, 223, 1009 (1969). 214. N i c h o l s , J.N., Nature, 225, 147 (1970). 215. L o d i s h , H.F., and Robertson, H.D., Cold Spring Harbor Sym. Quant. B i o l . , 34, 655 (1969). 216. S t e i t z , J.A., Dube, S.K., and Rudland, P.S., Nature, 226, 824 (1970). 217. D a r n e l l , J.E., B a c t e r i o l . Reviews, 3_2, 262 (1968). 218. Nirenberg, N.W., i n S.P. Colowick and N.O. Kaplan ( E d i t o r s ) , Methods i n Enzymology, V o l . V I , Academic P r e s s , New York, 1963, p. 17. 219. Iwasaki, K., S a b o l . , S., Wahba, A.J., and Ochoa, S., Arch. Biochem. Biophys., 125, 542 (1968). 220. Lucas-Lenard, J . , and Lipmann, F., Proc. Nat. Acad. S c i . U.S.A., 5_5, 1562 (1966). 221. Davis, B.J., Ann. N.Y. Acad. S c i . , 121, 404 (1964). 222. R i c h a r d s , E.G., C o l l , J.A., and G r a t z e r , W.B., A n a l . Biochem., 12, 452 (1965). 223. Peacock, A . C , and Dingman, C.W., B i o c h e m i s t r y , 6_, 1818 (1967) . 224. Peacock, A . C , and Dingman, C J . , B i o c h e m i s t r y , 1_, 668 (1968) . 158 225. Dingman, C.W., and Peacock., A . C, Bioc h e m i s t r y , 1_, 659 (1968) . 226. Cannon, M., Krug, R., and G i l b e r t , W., J . Mol. B i o l . , 7, 360 (1963). 227. Meselson, M., Nomura, M. , Brenner, S. , Davern, C , and S c h l e s s i n g e r , D. , J . Mol. B i o l . , 9_, 696 (1964) . 228. G i l l a m , I . , M i l l w a r d , S., Blew, D. von Tig e r s t r o m , M., Wimmer, E. , and Tener, 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., Bio c h e m i s t r y , 10, 889 (1971). 230. H a l b r e i c h , A., and Rabinowitz, M., Proc. Nat. Acad. S c i . U.S.A. , 68,' 294 (1971) . 231. Dunoff, J.S., and M a i t r a , U., Proc. Nat. 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., Proc. Nat. 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 . Mol. B i o l . , 54, 355 (1970). 235. Smith, A.E., and Marcker, K.A., Nature, 226, 607 (1970). 236. Brown, J.C., and Smith, A.E., Nature, 226, 610 (1970). 237. Caskey, C.T., Beaudet, A.L., and Nirenberg, M., J . Mol. B i o l . , 37, 99 (1968). 238. Gupta, N.K., J . B i o l . Chem., 243, 4959 (1968). 239. M a r s h a l l , R.E., Caskey, CT., and Nirenberg, M., Science, 155, 820 (1967). 240. Skogerson, L., and Moldave, K., Arch. Biochem. Biophys., 125, 497 (1968). 241. B l a c k , D.D., and G r i f f i n , A . C , Cancer Res., 3j0, 1281 (1970). 242. K l i n k , F., Kramer, G., Nour, A.M., and Petersen, K.G., Biochem. Biophys. A c t a , 134, 360 (1967). 243. R i c h t e r , D., and K l i n k , F., Bi o c h e m i s t r y , 6, 3569 (1967). 159 244. Fukami, H., and Imahori, K., Proc. Nat. Acad. S c i . U.S.A., 68, 570 (1971). 245. Lewin, B., Nature, 227, 1009 (1970). 246. G a l i b e r t , F. , L a r s e n , C. J . , L e l o n g , J . C , and B o i r o n , M., B u l l . Soc. Chim. B i o l . , £ 8 , 21 (1966). 247. Jordan, B.R., F o r g e t , B.G., and Monier, R., J . Mol. B i o l . , 55, 407 (1971). 248. J o r d a n , R.R., J . Mol. B i o l . , 5_5, 423 (1971). 249. L e v i n , J.G., and N i r e n b e r g , M., J . Mol. B i o l . , 34, 467 (1968). 250. L e v i n , J.G., J . B i o l . Chem., 245, 3195 (1970). 251. Hoagland, M.B., and Coonly, L.T., Proc. Nat. Acad. S c i . U.S.A., 46, 1554 (1960). 252. Takanami, M. , Biochim. Biophys. A c t a , 55_, 132 (1962). 253. Yu, C.T., and Zamecnik, P . C , Biochim. Biophys. A c t a , 45, 148 (1960). 254. P r e i s s , J . , Dieckmann, M., and Berg, P., J . B i o l . Chem., 236, 1748 (1961). 255. N i r e n b e r g , M., and Leder, P., S c i e n c e , 145, 1399 (1964). 256. Hardesty, B., A r l i n g h a u s , R., S h a e f f e r , J . , and Schweet, R. , C o l d S p r i n g Harbor Sym. Quant. 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 . , and Ochoa, S., Proc. Nat. Acad. S c i . U.S.A., 5£, 290 (1966). 258. Ofengand, J . , and Hines, C., J . B i o l . Chem., 244, 6241 (1969) . 259. Shimizu, N., Hayashi, H., and Muira, K., J . Biochem. (Tokyo) , £ 7 , 373 (1970) . 260. Warner, J.R., and R i c h , A., Proc. Nat. Acad. S c i . U.S.A., 51, 1134 (1964). 261. Takanami, M., Biochim. Biophys. A c t a , 61, 432 (1962). 262. W e t t s t e i n , F.O., and N o l l , H., J . Mol. B i o l . , 11, 35 (1965). 263. G i l b e r t , W., J . Mol. B i o l . , 6, 389 (1963). 160 264. E l s o n , D. , Biochim. Biophys. A c t a , 53_, 232 (1961). 265. E l s o n , D., Biochim. Biophys. A c t a , 61, 460 (1962). 266. Oppenheim, J . , Scheinbuks, J . , B i a v a , C., and Marcus, L., Biochim. Biophys. A c t a , 161, 386 (1968). 267. Gonzalez, N.S., Goldenberg, S.H., and A l g r a n a t i , I.D., Biophys. A c t a , 166, 760 (1968). 268. Scheps, R., Wax, R., and R e v e l , M., Biochim. Biophys. A c t a , 232, 140 (1971). 269. I g a r a s h i , K., and K a j i , A., Eur. J . Biochem., 14, 41 (1970). 270. von D i g g e l e n , O.P., H e i n s i u s , H.L., Kalousek, F., and Bosch, L., J . Mol. B i o l . , 55, 277 (1971). 271. S c h r e i e r , N.H., and N o l l , H., Nature, 229, 128 (1970). 272. G i l b e r t , W., J . Mol. B i o l . , 6, 389 (1963). 273. M o s t e l l e r , R.D., Cul p , W.J., and Hardesty, B., J . B i o l . Chem., 243, 6343 (1968). 274. Muench, K.H., and Berg, P., B i o c h e m i s t r y , 5_, 970 (1966). 275. Muench, K.H., and S a f i l l e , P.A., B i o c h e m i s t r y , 1_, 2799 (1968) . 276. C u l p , W., M o r r i s e y , J . , and Hardesty, B., Biochem. Biophys. Res. Commun., 4£, 777 (1970). 277. Smith, D.W.E., and McNamara, A.L., S c i e n c e , 171, 577 (1971). 278. Burkard, G. , G u i l l e m a n t , P., and W e i l , J.H., Biochim. Biophys. A c t a , 224, 184 (1970). 279. Muench, K.H., and Berg, P., i n G.L. C a n t o n i and D.R. Davies ( E d i t o r s ) , Procedures i n N u c l e i c A c i d Research, Harper and Row, New York, 1966, p. 375. 280. Shearn, A., and Horowitz, 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., and Wahba, A . J . , i n L.Grossman and K. Moldave ( E d i t o r s ) , Methods i n Enzymology, V o l . X I I , Academic P r e s s , New York, 196 7, p. 524. 1 6 1 2 8 2 . H i l l , W.E., Anderegg, J.W., and yan Holde, K.E., J. Mol. Bio l . , 5 3 , 1 0 7 ( 1 9 7 0 ) . 2 8 3 . Bock, R.M., and Cherayil, J.D., in L. Grossman and K. Moldave (Editors), Methods in Enzymology, Vol. XII, Academic Press, New York, 1 9 6 7 , p. 6 3 8 . 2 8 4 . Heppel, L.A., i b i d . , Vol. XII, 1 9 6 7 , p. 3 1 6 . 2 8 5 . Kuriki, Y. , and Kaji, A., J. Mol. Bio l . , 2_5, 4 0 7 ( 1 9 6 7 ) . 2 8 6 . Comb, D..G. , and Zehavi-Willner, T. , J. Mol. B i o l . , 2 3 , 4 4 1 ( 1 9 6 7 ) . 2 8 7 . Sarkar, N., and Comb, D.G., J. Mol. B i o l . , 39_, 3 1 ( 1 9 6 9 ) . 2 8 8 . Comb, D.G. , and Sarkar, N. , J. Mol. Bio l . , 2 5 _ , 3 1 7 ( 1 9 6 7 ) . 2 8 9 . Reynier, M., and Monier, R., Bull. Soc. Chim. B i o l . , 5 0 , 1 5 8 3 ( 1 9 6 8 ) . 2 9 0 . Nirenberg, M., and Leder, P., Science, 1 4 5 , 1 3 9 9 ( 1 9 6 4 ) . 2 9 1 . Bartz, J., S o i l , D., Burrows, W.J., and Skoog, F., Proc. Nat. Acad. Sci. U.S.A., 6 7 , 1 4 4 8 ( 1 9 7 0 ) . 2 9 2 . Roy, K.L., Bloom, A., and S o i l , D., preprint. 2 9 3 . Gillam, I.C., and Tener, G.M., in L. Grossman and K. Moldave (Editors), Methods in Enzymology, Vol. XX, Part C, Academic Press, New York, 1 9 7 1 , p. 5 5 . 2 9 4 . Stephenson, M.L., and Zamecnik, P.C., in L. Grossman and K. Moldave (Editors), Methods in Enzymology, Vol. XII, Part A, Academic Press, New York, 1 9 6 7 , p. 6 7 0 . 2 9 5 . Roy, K.L., and S o l i , D., Biochem. Biophys. Acta, 1 6 1 , 5 7 2 ( 1 9 6 8 ) . 2 9 6 . Jardetsky, O., and Julian, G.R., Nature, 2 0 1 , 3 9 7 ( 1 9 6 4 ) . 2 9 7 . Spirin, A.S., Kiselev, N.A., Shakulov, R.S., and Bogdanov, A.A., Biokhimiya, 28, 7 6 5 ( 1 9 6 3 ) . 2 9 8 . Elson, D., in D.B. Roodyn (Editor), Enzyme Cytology, Academic Press, New York, 1 9 6 7 , p. 4 0 7 . 2 9 9 . Szer, W. , Biochem. Biophys. Res. Commun., 35_ 6 5 3 ( 1 9 6 9 ) . 3 0 0 . Egami, F., and Nakamura, K., in A. Kleinzeller, G.F. Springer and H.G. Wittmann (Editors), Molecular Biology, Vol. 6 , Academic Press, New York, 1 9 6 9 . 162 301. Neu, H.C, and Heppel, L.A. , J . B i o l . Chem., 239, 3893 (1964). 302. D e l i h a s , N. , Biochem. Biophys. Res. Commun., 3_9, 905 (1970) . 303. Ehresmann, C., and E b e l , J.P., Eur. J . Biochem., 13, 577 (1970). 304. Gupta, S.L., Chen, J . , Schaefer, L., Lengyel, P., and Weissman, S.M., Biochem. Biophys. Res. Commun., 39, 883 (1970). 305. Kuechler, E., and R i c h , A., Nature, 225, 920 (1970). 306. Kurland, C.G., J . Mol. B i o l . , 18, 90 (1966). 307. C r i c k , F.H.C, J . Mol. B i o l . , 19, 548 (1966). 308. Dixon, G.H., pe r s o n a l communication. 309. Gilmour, S., and Dixon, G.H., perso n a l communication. 163 APPENDIX 50 y l o f ^C-Phe (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 water. 5 y l of t h i s mixture was used f o r each assay. Two c o n t r o l s were used throughout. One con-t r o l contained a l l the assay components except polyU w h i l e the other c o n t r o l contained a l l the assay components except WRib. The l e v e l of r a d i o a c t i v i t y observed i n both c o n t r o l s was the same—approximately 200 cpm. This l e v e l of r a d i o -a c t i v i t y served as the background and was s e t at one. 50 y l of 1'*C-amino a c i d mixture (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 water. 5 y l of t h i s d i l u t e d mixture was used f o r each assay. Enzyme prepared as d e s c r i b e d on pages 52-53 was added. A l l tubes contained the same amount of tRNA. Two c o n t r o l s were used throughout. One c o n t r o l con-t a i n e d a l l the assay components except tRNA (tRNA c o n t r o l ) w h i l e the o t h e r c o n t r o l contained a l l the assay components except enzyme. Since the tRNA c o n t r o l gave the h i g h e s t background 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 of the sample tubes. The assay system contained a l l the 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. A l l the tubes contained the same amount of tRNA. Enzyme prepared as d e s c r i b e d on pages 52-53 was used : 5 y l of the s p e c i f i c 1''C-amino a c i d was added t o the p a r t i c u l a r tube. 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 of the 1 "'C-amino acids 164 used are given below: pg/ml mCi/ml A l a 76.0 0 .10 Arg 37.0 0 .05 Asn 282.0 0 .10 Asp 86 .0 0 .10 Gly 56 .0 0 .10 Glu 71.0 0 .10 His 1.2 0.05 H e 55.0 0.10 Leu 52.0 0 .10 Lys 30 .0 0 .05 Met 1100 .0 0 .10 Phe 42.0 0 .10 Pro 31.0 0 .05 Ser 84.0 0 .10 Thr 37.0 0.05 Trp 900 .0 0 .10 Tyr 48.0 0 .10 V a l 63.0 0 .10 Two c o n t r o l s were used 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 contained a l l the assay components except tRNA (tRNA c o n t r o l ) w h i l e the other c o n t r o l contained a l l the assay components except enzyme. Since the tRNA c o n t r o l gave the h i g h e s t background l e v e l of r a d i o a c t i v i t y , t h i s background was s u b t r a c t e d from the sample tube c o n t a i n i n g tRNA. The experimental c o n d i t i o n s are s i m i l a r t o those used by K e l l e r (229) and Muench and Berg (279). 

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