UBC Theses and Dissertations

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UBC Theses and Dissertations

A study of tRNA biosynthesis in Escherichia coli Chase, Randal 1974

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A STUDY OF tRNA BIOSYNTHESIS I N E s c h e r i c h i a c o l i b y RANDAL.CHASE B . S c , B i s h o p ' s U n i v e r s i t y , 1970 A THESIS SUBMITTED I N PAR T I A L FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY i n t h e D e p a r t m e n t o f B i o c h e m i s t r y We a c c e p t t h i s t h e s i s a s c o n f o r m i n q t o t h e r e q u i r e d s t a n d a r d THE UNIVERSITY OF B R I T I S H COLUMBIA S e p t e m b e r , 1974 In presenting th is thesis in pa r t i a l fu l f i lment of the requirements for an advanced degree at the Univers i ty of B r i t i s h Columbia, I agree that the L ibrary shal l make it f ree ly ava i lab le for reference and study. I further agree that permission for extensive copying of this thesis for scholar ly purposes may be granted by the Head of my Department or by his representat ives. It is understood that copying or pub l i cat ion of this thesis for f inanc ia l gain sha l l not be allowed without my written permission. Department of The Univers i ty of B r i t i s h Columbia Vancouver 8, Canada Date (\ 'so m y ABSTRACT E s c h e r i c h i a c o l i was g r o w n i n t h e p r e s e n c e o f amino a c i d a n a l o g u e s o r i n t h e a b s e n c e o f r e q u i r e d amino a c i d s . The tRNAs. w e r e i s o l a t e d a n d c h a r a c t e r i z e d . Numerous c h a n g e s w e r e o b s e r v e d i n t h e t o t a l tRNA a c c e p t a n c e f o r p a r t i c u l a r a m i n o a c i d s a l t h o u g h i n no i n s t a n c e d i d t h e s e c h a n g e s o c c u r f o r a m i n o a c i d s c o r r e s -p o n d i n g t o t h e a d v e r s e g r o w t h c o n d i t i o n . The i s o a c c e p t o r p a t t e r n s f o r p a r t i c u l a r l a b e l l e d a m i n o a c y l - t R N A s w e r e d e t e r m i n e d o n t h e a n i o n e x c h a n g e r RPC-5. N o v e l i s o a c c e p t o r t R N A s w e r e o b -s e r v e d u n d e r s e v e r a l g r o w t h c o n d i t i o n s . S i g n i f i c a n t c h a n g e s i n tRNA i s o a c c e p t o r d i s t r i b u t i o n s w e r e n o t e d . I n c e r t a i n i n s t a n c e s i t a p p e a r e d t h a t c h a n g e s i n t o t a l a m i no a c i d a c c e p -t a n c e c o u l d be e x p l a i n e d i n t e r m s o f t h e i n c r e a s e d o r d e c r e a s e d s y n t h e s i s o f p a r t i c u l a r tRNA i s o a c c e p t o r s w h i l e f o r o t h e r tRNAs i t seemed t h a t c h a n g e s o c c u r r e d i n t h e s y n t h e s i s o f a l l i s o -a c c e p t o r s f o r a p a r t i c u l a r a m ino a c i d s u c h t h a t t h e r e l a t i v e a m ounts o f i s o a c c e p t o r s r e m a i n e d c o n s t a n t e v e n when t o t a l a m i no a c i d a c c e p t a n c e c h a n g e d c o n s i d e r a b l y . E. c o l i was grown o v e r a w i d e t e m p e r a t u r e r a n g e , 17°C t o 44°C, a n d t h e tRNA i s o l a t e d a n d c h a r a c t e r i z e d . N o v e l tRNA i s o -a c c e p t o r s w e r e o b s e r v e d a t b o t h h i g h a n d l o w g r o w t h t e m p e r a t u r e s f o r m o s t b u t n o t a l l t R N A s . I t was shown t h a t t h e same i s o -a c c e p t o r s c o u l d be f o r m e d a t b o t h e x t r e m e s o f t e m p e r a t u r e . P r e -l i m i n a r y r e s u l t s s u g g e s t t h a t t h e n o v e l i s o a c c e p t o r s a r e f o r m e d a s t h e r e s u l t o f a t e m p e r a t u r e a g g r a v a t i o n o f a n u t r i t i o n a l p r o b l e m a t e x t r e m e s o f g r o w t h t e m p e r a t u r e . One of the novel tRNA isoacceptors formed under a variety Val of adverse growth conditions, tRNA3 , was p u r i f i e d and p a r t i a l l y Val characterized. The results are consistent with tRNA3 being Val an undermodified precursor of the major isoacceptor tRNA^ E. c o l i s t r D was grown and the tRNA is o l a t e d and character-ized. Major differences i n the amino acid acceptances for several tRNAs were observed. These changes were accomplished without any s i g n i f i c a n t changes i n the r e l a t i v e isoacceptor d i s t r i b u t i o n s as determined by RPC-5 chromatography. Gel electrophoretic analysis was performed on tRNA from c e l l s grown at extremes of growth temperature. S i g n i f i c a n t d i f f e r -ences were observed i n the 5S region; there was an accumulation of material i n c e l l s grown at low temperature and a decrease of material i n c e l l s grown at high temperature. i i i . TABLE OF CONTENTS Page ABSTRACT . . . . . . . i TABLE OF CONTENTS i i i LIST OF TABLES v LIST OF FIGURES vi ACKNOWLEDGEMENTS ix DEDICATION x ABBREVIATIONS xi INTRODUCTION 1 tRNA Genes i n E. c o l i 1 tRNA Transcription 2 The 3' OH Ends of tRNA 6 The Stringent Response 8 Sp e c i f i c Control of tRNA Synthesis . 11 Functional Adaptation 12 tRNA During Differentation and Development 15 Hormonal Ef f e c t s on tRNA 18 Organ and Tissue tRNA Differences 19 tRNA and Phage Infection 20 tRNA and Cancer 21 tRNA and Growth Conditions 22 Minor Nucleosides i n tRNA 26 MATERIALS 33 METHODS 35 Growth of E. c o l i 35 iv. Page Preparation of Aminoacyl-tRNA Synthetases 36 Preparation of tRNA . . 37 Aminoacylation of tRNA;Acceptance Levels . . . . . . 37 Aminoacylation of tRNA for RPC-5 Chromatography . . . 38 RPC-5 Chromatography 39 P u r i f i c a t i o n of tRNA* 39 Polyacrylamide Gel Electrophoresis . . 41 Nucleoside Analysis of P u r i f i e d tRNAs . . . . . . . . 42 Qualitative Nucleotide Analysis of tRNA^ 1 43 [ 3H]Val-Oligonucleotides on DEAE-Cellulose 43 RESULTS AND DISCUSSION 44 Amino Acid Analogues 44 tRNA of E. c o l i Treated with Amino Acid Analogues and of E. c o l i Depleted of Es s e n t i a l Amino Acids . 47 RPC-5 Chromatography 54 E f f e c t of Growth Temperature on E. c o l i tRNA . . . . 86 Val Characterization of tRNA^ 117 P u r i f i c a t i o n of tRNA^ 1 1 121 Spectral Char a c t e r i s t i c s of tRNA^ a''" . 123 Qualitative Nucleotide Analysis of tRNA^ a l, 123 Val Randerath Nucleoside Analysis of tRNA^ 127 tRNA of E. c o l i B s t r D 131 Gel Electrophoresis of Crude E. c o l i tRNA . . . . . . 138 CONCLUSIONS 143 BIBLIOGRAPHY 145 V. LIST OF TABLES Page Table 1. E f f e c t of amino, acid analogues on E. c o l i growth 45 Table 2. Amino acid acceptance of crude tRNA from E. c o l i grown i n the presence of amino acid analogues 49 Table 3. Amino acid acceptance of crude tRNA of E. c o l i NF162 c e l l s depleted of an esse n t i a l amino acid 50 Table 4. Amino acid acceptance of crude tRNA of E. c o l i B c e l l s grown at various temperatures 87 Table 5. Amino acid acceptance of crude tRNA of E. c o l i B c e l l s grown at high temperature 113 Table 6. Nucleoside analysis of tRNA V a l 129 Table 7. Amino acid acceptance of crude tRNA of E. c o l i B s t r D 132 vi. LIST OF FIGURES Page Figure 1. RPC-5 chromatography of [ 3H]Val-tRNA V a l (B) . . 57 Figure 2. RPC-5 chromatography of [ 3H]Val-tRNA (B-tyrosine phosphonate) . . . . . . . . 57 Val Figure 3. RPC-5 chromatography of [ 3H]Val-tRNA (B; p-fluorophenylalanine) 60 Figure 4. RPC-5 chromatography of [. 3H] Val-tRNA (B-stationary state) 60 Figure 5. RPC-5 chromatography of [ 3H]Val-tRNA V a l (NF162) 63 Veil Figure 6. RPC-5 chromatography of [ 3H]Val-tRNA (NF162; O-methyl-DL-threonine) . . 63 Veil Figure 7. RPC-5 chromatography of [ 3H]Val-tRNA (NF162; a-amino-n-butyrate) . . . . . . . . . . 65 Figure 8. Cochromatography on RPC-5 of [ 1 4C]Val-tRNA V a l (B) and [ 3H]Val-tRNA V a l (NF162-Arg) 65 Figure 9. Cochromatography on RPC-5 of [ 1 1 1C] Val-tRNA (B) and [ 3H]Val-tRNA V a l (NF162-Met) 68 ^ Val Figure 10. Cochromatography on RPC-5 of [ H]Val-tRNA (NF162-Arg) and [;1 ^ ClVal-tRNAVal (NF162-Met) . . 68 Figure 11. RPC-5 chromatography of [ 1^C]Ser-tRNA b (B). . 71 Figure 12. RPC-5 chromatography of [ 1"dSer-tRNA S e r (B-tyrosine phosphonate) 71 Figure 13. RPC-5 chromatography of [ 3H]Ser-tRNA S e r (NE162) 74 Figure 14. RPC-5 chromatography of [ 3H]Ser-tRNA S e r (NF162; a-amino-n-butyrate) 74 Ser Figure 15. RPC-5 chromatography of [ H]Ser-tRNA (NF162-Arg) 75 Figure 16. RPC-5 chromatography of [ 1^C]Leu-tRNA L e u (NF162) 77 Figure 17. Cochromatography on RPC-5 of [ 3H]Leu-tRNA L e u (NF162; a-amino-n-butyrate) and [ 1 1*C]Leu-tRNA^eu (B) . . . . . . . . . . . . 78 v i i . Page Figure 18. Cochromatography on RPC-5 of [ 3H]Leu-tRNA L e u (NF162-Arg) and [1*C]Leu-tRNALeu (B) 79 Figure 19. Cochromatography on RPC-5 of [ 1 4C]Val-tRNA V a l (B-37°) and rH]Val-tRNA V a l (B-44°) . . . . . . 90 Figure 20. Cochromatography on RPC-5 of [ 1 4C]Val-tRNA V a l (B-44°) and rH]Val-tRNA V a l (NF162-Arg) . . . . 90 Figure 21. RPC-5 chromatography of [ 3H]Val-tRNA V a l (B-41°) 91 Figure 22. RPC-5 chromatography of [ 3H]Val-tRNA V a l (B-30°) 92 Figure 23. Cochromatography on RPC-5 of [ 1"C]Val-tRNA V a l (B-37°) and [*H]Val-tRNA V a l (B-20°) 95 Figure 24. Cochromatography on RPC-5 of [ 1^C]Val-tRNA V a l (B-20°) and pH]Val-tRNA V a l (NF162-Arg) . . . . 95 Figure 25. Cochromatography on RPC-5 of [ 1 hC]Val-tRNA V a l (B-37°) and [^H]Val-tRNAVal (B-17°) 97 Figure 26. Cochromatography on RPC-5 of [ 1^C]Val-tRNA V a l (B-17°) and rH] Val-tRNA V al (NF162-Arg) . . . . 97 Figure 27. RPC-5 chromatography of [ 1 "*C] Leu-tRNA L e u (B-17°) 99 Figure 28. RPC-5 chromatography of [ 1^C]Leu-tRNA L e u (B-20°) 100 Figure 29. RPC-5 chromatography of [ 1^C]Leu-tRNA L e u (B-44°) 101 Figure 30. RPC-5 chromatography of [ H]Ser-tRNA (B-17°) 102 Figure 31. RPC-5 chromatography of [ 3H]Ser-tRNA S e r (B-44°) 103 Figure 32. Cochromatography on RPC-5 of [ 1 **C] Thr-tRNA T h r (B-37°) and [*H]Thr-tRNA T h r (B-17°) 104 Figure 33. Cochromatography on RPC-5 of [ 1^C]Thr-tRNA (B-37°) and [*H]Thr-tRNA T h r (B-20°) 105 v i i i . Page Figure 34. Cochromatography on RPC-5 of [ 1 hC] Thr-tRNA T h r (B-37°) and [ H]Thr-tRNA (B-44°) . . . . . . 106 Figure 35. RPC-5 chromatography of [ 3H]Val-tRNA V a l (B-12°) Flask grown c e l l s 109 Figure 36. RPC-5 chromatography of [. 3H] Val-tRNA (B-41.5°) Flask grown c e l l s 109 T T - a "j Figure 37. RPC-5 chromatography of [ 3H]Val-tRNA (B-42°) Flask grown c e l l s 112 Figure 38. RPC-5 chromatography of [ 3H]Val-tRNA V a l (W-21°) 112 Figure 39. [ 3H]Val-RNase T± oligonucleotides on DEAE-ce l l u l o s e 120 Figure 40. RPC-5 chromatography of tRNA^ 1 . 122 Figure 41. U l t r a v i o l e t absorption spectra of E. c o l i tRNA 125 Val Figure 42. Nucleotide analysis of tRNA^ 126 Figure 43. Randerath nucleoside analysis of tRNAY a l and tRNA V a l . 128 Figure 44. RPC-5 chromatography of [ 3H]Val-tRNA (B strD) 134 Figure 45. RPC-5 chromatography of [ 1 ""C] Leu-tRNA L e u (B strD) 135 3 S 61* Figure 46. RPC-5 chromatography of [ H]Ser-tRNA (B s t r D ) 13 6 Figure 47. Acrylamide gel electrophoresis of crude tRNA preparations of E. c o l i B grown at several d i f -ferent temperatures 139 Figure 48. Acrylamide gel electrophoresis of crude tRNA preparations of E. c o l i grown at d i f f e r e n t temperatures .140 Figure 49. Acrylamide gel electrophoresis of crude tRNA preparations of E. c o l i grown at d i f f e r e n t temp-eratures , 141 ix. A c k n o w l e d g e m e n t s F i r s t a n d f o r e m o s t I ' d l i k e t o t h a n k my r e s e a r c h s u p e r v i s o r D r . G.M. T e n e r f o r h i s e n d l e s s e n c o u r a g e m e n t a nd g u i d a n c e d u r i n g t h e c o u r s e o f t h i s w o r k . I h a v e e n j o y e d v e r y much w o r k i n g i n h i s l a b o r a t o r y a n d f e e l t h a t i t h a s b e e n a v e r y r i c h a n d r e w a r d -i n g e x p e r i e n c e f o r me. I am d e e p l y i n d e b t e d t o D r . I a n G i l l a m , a s a r e a l l g r a d u a t e s t u d e n t s who h a v e w o r k e d i n t h i s l a b o r a t o r y , f o r v a l u a b l e d i s -c u s s i o n s a n d f o r h i s w i l l i n g n e s s t o t a k e t h e t i m e t o t e a c h p r o p e r l a b o r a t o r y t e c h n i q u e s . I t i s my p l e a s u r e t o a c k n o w l e d g e t h a t t h e g e l e l e c t r o p h o r e s i s e x p e r i m e n t s d e s c r i b e d i n t h i s t h e s i s w e r e p e r f o r m e d b y D r . I a n G i l l a m . I w o u l d l i k e t o t h a n k D r . B r a d l e y W h i t e f o r m a t e r i a l s a n d f o r many h o u r s o f v a l u a b l e d i s -c u s s i o n s p a r t i c u l a r l y r e g a r d i n g e x p e r i m e n t a l a p p r o a c h a n d t e c h -n i q u e . Many o t h e r p e o p l e , b y t h e i r t h o u g h t f u l d i s c u s s i o n , h a v e h e l p e d me. I n p a r t i c u l a r , I w o u l d l i k e t o t h a n k D r s . D a v i d B a i l l i e , M i l d r e d C o h n , M i c h a e l S m i t h , R o b e r t W a r r i n g t o n , J o h n G a l l a n t a n d W.J. P o l g l a s e . I was t h e f o r t u n a t e r e c i p i e n t o f a N a t i o n a l R e s e a r c h C o u n c i l "1967 S c i e n c e S c h o l a r s h i p " f o r t h e p e r i o d J u n e 1970 t o J u n e 1974 a n d h a v e s i n c e b e e n s u p p o r t e d b y a M e d i c a l R e s e a r c h C o u n c i l g r a n t t o D r . G.M. T e n e r . I g r a t e f u l l y a c k n o w l e d g e t h e s u p p o r t I h a v e r e c e i v e d . X . DEDICATION to June, C a r r i e and David XI, . ABBREVIATIONS USED The a b b r e v i a t i o n s s u m m a r i z e d b e l o w a r e t h o s e s u g g e s t e d by t h e IUPAC-IUB C o m b i n e d C o m m i s s i o n o n B i o c h e m i c a l N o m e n c l a t u r e [ R e v i s e d T e n t a t i v e R u l e s , 1965 (261) a n d R e c o m m e n d a t i o n s , 197 0 ( 2 6 2 ) ] . R e c o m m e n d a t i o n s (1970) (262) r e p l a c e S e c t i o n 5 o f t h e R e v i s e d T e n t a t i v e R u l e s (1965) ( 2 6 1 ) . A, C, G, U: -Ap, Cp, G p f a n d Up: -GDP: -GTP: -ATP: -pppGp: -ppGpp: -pppGpp: -p o l y ( C , G,A): -DNA: -RNA: -rRNA: = mRNA: -tRNA: -tRNA V a l t h e r i b o n u c l e o s i d e s o f t h e f o u r b a s e s ; a d e n i n e , c y t o s i n e , g u a n i n e , and u r a c i l . t h e 3 ' - r i b o n u c l e o s i d e monophos-p h a t e s t h e 5 ' - r i b o n u c l e o s i d e d i p h o s p h a t e o f g u a n o s i n e t h e 5 ' - r i b o n u c l e o s i d e t r i p h o s p h a t e o f g u a n o s i n e t h e 5 ' - r i b o n u c l e o s i d e t r i p h o s p h a t e o f a d e n o s i n e t h e r i b o n u c l e o s i d e - 5 ' - t r i p h o s p h a t e -3 *-monophosphate o f g u a n o s i n e t h e r i b o n u c l e o s i d e - 5 * - d i p h o s p h a t e -3 1 - d i p h o s p h a t e o f g u a n o s i n e t h e r i b o n u c l e o s i d e - 5 * - t r i p h o s p h a t e 3 ' - d i p h o s p h a t e o f g u a n o s i n e p o l y r i b o n u c l e o t i d e w i t h a random s e q u e n c e c o n t a i n i n g C, G a n d A d e o x y r i b o n u c l e i c a c i d r i b o n u c l e i c a c i d r i b o s o m a l r i b o n u c l e i c a c i d m e s s e n g e r r i b o n u c l e i c a c i d t r a n s f e r r i b o n u c l e i c a c i d n o n a c y l a t e d v a l i n e tRNA x i i . Val-tRNA : -tRNAY a l: -Val, Ser, Leu, Thr: SDS: -EDTA: -RPC: -DEAE-cellulose: -BD-cellulose: -A 2 6 o : -A 2 6 0 u n i t : -RNase: -TuTs: -G-factor: -. D s t r : -d.p.h.: -aminoacylated valine tRNA one of the isoaccepting species of tRNA V a l amino acids, valine, serine, leucine, threonine sodium dodecyl sulfate ethylenediaminetetraacetate reverse phase chromatography 0-(diethylaminoethyl) c e l l u l o s e benzoylated DEAE-cellulose absorbance at 260 nm the amount of material giving an absorbance of 1.0 i n 1.0 ml of solution at neutral pH i n a 1 cm l i g h t path at 260 nm. ribonuclease transfer factors i n protein synthesis translocation factor a mutant form that i s dependent upon streptomycin for growth growth rate expressed i n terms of doublings of t u r b i d i t y per hour at 420 nm 1. INTRODUCTION The manner i n which tRNA i s involved i n the complex process of protein synthesis has been the subject of numerous reviews (1-8). Physical and chemical characterization of tRNA (4, 9-13) has likewise been reviewed i n great d e t a i l . The introduction to t h i s thesis w i l l therefore not attempt to cover a l l aspects of present day knowledge of tRNA structure and function but w i l l attempt to bring together and summarize the important informa-t i o n on tRNA t r a n s c r i p t i o n , processing of the primary trans-c r i p t i o n a l product and f i n a l l y the maturation of the tRNA par-t i c u l a r l y with respect to modified nucleoside composition. Emphasis w i l l be placed on known and postulated control mech-anisms for the synthesis and maturation of tRNA. A number of review a r t i c l e s are available which deal with various aspects of these topics (14-25, 137). tRNA Genes i n E. c o l i Hybridization experiments have suggested 40 to 8 0 l o c i for tRNA genes i n Escherichia c o l i (20) and primarily from studies of missense and nonsense suppressor tRNA mutations, i t has been shown that these tRNA genes are widely d i s t r i b u t e d around the E. c o l i chromosome (20,23). Evidence has been put forward, however, that suggests that tRNA genes are usually found i n cl u s t e r s . Squires et a l . (26) showed that gly T s u + 3 6 (a mutant form of tRNA^1^) , tRNA^^ and tRNA^*137 genes are i n very close proximity. Squires and Carbon (27) e a r l i e r presented evidence for the existence of several c l o s e l y linked i d e n t i c a l 2. c o p i e s o f t h e g e n e s f o r tRNA^ *. ; O h l s s o n e t a l . (28) s u g g e s t t h a t t h e o c h r e s u p p r e s s o r su_, a n d t h e amber s u p p r e s s o r s u I I a r e 50 t o 150 b a s e p a i r s f r o m e a c h o t h e r . t R N A ^ y r h a s b e e n shown t o e x i s t a s two i d e n t i c a l t a n d e m g e n e s ( 2 9 ) . O r i a s e t a l . (30) d e m o n s t r a t e d t h a t g l y T s u + 3 6 a n d t h e o c h r e s u p p r e s s o r s u p l 5 B (supM) a r e c l o s e l y l i n k e d . T h i s i s p r o b a b l y t h e same r e s u l t o b t a i n e d by S q u i r e s e t a l . (26) i n t h a t s u p l 5 B . i n s e r t s t y r o s i n e . U n p u b l i s h e d w o r k (31) s u g g e s t s t h a t i n v i v o E. c o l i tRNA t r a n s -c r i p t s may o f t e n be up t o f i v e t RNAs l o n g s i n c e i n one i n s t a n c e t h e y o b s e r v e d a t r a n s c r i p t c o n t a i n i n g f i v e c o v a l e n t l y l i n k e d tRNA m o l e c u l e s . T r a n s c r i p t s h a v i n g s i n g l e c o p i e s o f s e v e r a l d i f f e r e n t tRNA g e n e s ( s i m i l a r t o t h a t d e s c r i b e d by S q u i r e s e t a l . ( 3 1 ) ) w e r e a l s o o b s e r v e d . S e v e r a l a u t h o r s (32,33) i n t h e c o u r s e o f i s o l a t i n g tRNA g e n e s h a v e l i k e w i s e s u g g e s t e d t h a t tRNA c i s t r o n s may be c l u s t e r e d i n g r o u p s o f two o r t h r e e . tRNA T r a n s c r i p t i o n I n E. c o l i , tRNA g e n e s a r e t r a n s c r i b e d b y a n RNA p o l y m e r a s e c o m p l e x i n t h e p r e s e n c e o f t h e r e q u i r e d n u c l e o s i d e t r i p h o s p h a t e s a n d a p p r o p r i a t e c o - f a c t o r s . The i n i t i a l t r a n s c r i p t i o n p r o d u c t i s l a r g e r t h a n m a t u r e tRNA a n d c o n s t i t u t e s a p r e c u r s o r w h i c h i s m a t u r e d by s e l e c t i v e n u c l e a s e a c t i o n and m i n o r b a s e m o d i f i c a t i o n . The s i z e o f t h e p r i m a r y t r a n s c r i p t i o n a l p r o d u c t h a s b e e n t h e s u b j e c t o f c o n s i d e r a b l e r e s e a r c h . K i n e t i c s t u d i e s b y V i c k e r s and M i d g l e y (34) s u g g e s t t h a t t h e a v e r a g e tRNA p r e c u r s o r i n E. c o l i m i g h t be 17 0 ± 40 n u c l e o -t i d e s l o n g . I n d i v i d u a l p r e c u r s o r t R N A s o r p r e - t R N A m o l e c u l e s 3. have been studied. In p a r t i c u l a r , Altman and co-workers (35, 36) have used a 0 80 phage, carrying mutations i n tRNAg^ + +, to Tvr cause the accumulation of precursor tRNA g^ + + +. Presumably pro-cessing i s slower because the maturation enzymes do not have the same a f f i n i t y for the mutant substrate. The t o t a l nucleotide sequence of the precursor was determined and found to have a 5'-terminal pppGp, suggesting natural i n i t i a t i o n , followed by an additional 40 nucleotides and then the nucleotide sequence which i s eventually converted into tRNAg^ + +. Ribosome-associated RNase P was shown to remove s e l e c t i v e l y the extra 41 nucleotides at the 5'end (22,37). The 3'OH end of the precursor was three nucleotides longer than the mature molecule and had the com-pleted pCpCpA end c h a r a c t e r i s t i c of mature tRNA. Primakoff and Schedl (31) have shown i n unpublished work that the 3'OH end of the Altman precursor i s shorter than the true precursor. Loewen et a l . (305) have recently determined the sequence of 23 nucleotides beyond the CCA end. Primakoff and Schedl confirm Altman's re s u l t s regarding the extra nucleotides at the 5'end and report that four s p e c i f i c endonucleases are normally i n -volved i n the maturation of the precursor. Two s p e c i f i c cleav-ages occur at each end. They postulate that cleavage by RNase P, l i b e r a t i n g the 41 nucleotide fragment, i s prerequisite for the other three cleavages. The 41 nucleotide fragment i s cleaved s p e c i f i c a l l y by a second nuclease. One might ask why the cleaved 41 nucleotide fragment undergoes a second highly s p e c i f i c cleavage and i s not just simply degraded by a general exonuclease. 4. D i j k a n d S i n g h a l (38) h a v e r e c e n t l y r e p o r t e d t h e a c c u m u l a -t i o n o f p r e - t R N A i n E. c o l i e i t h e r s t a r v e d f o r m e t h i o n i n e o r i n h i b i t e d b y c h l o r a m p h e n i c o l . By S e p h a d e x c h r o m a t o g r a p h y t h e y d i s t i n g u i s h e d a g r o u p o f p r e c u r s o r s w h i c h t h e y c a l l ex's, w h i c h a r e a b o u t 200 n u c l e o t i d e s l o n g , a n d a s e c o n d g r o u p c a l l e d $'s, a b o u t 120 n u c l e o t i d e s l o n g . B o t h g r o u p s a r e v e r y h e t e r o g e n e o u s when c h r o m a t o g r a p h e d on RPC-5. B o t h g r o u p s c a n be c l e a v e d t o 4S m a t e r i a l b y c r u d e c e l l e x t r a c t s a n d h a v e s e q u e n c e h o m o l o g y w i t h tRNA a s d e t e r m i n e d by c o m p e t i t i v e h y b r i d i z a t i o n . S i m i l a r l y , b a n d s C, D a n d F' o b s e r v e d b y g e l e l e c t r o p h o r e s i s b y G r i f f i n a n d B a i l l i e (39) a r e tRNA p r e c u r s o r s t r a n s i e n t l y a c c u m u l a t e d u p o n t h e r e l i e f o f g r o w t h r e s t r i c t i n g c o n d i t i o n s . U n p u b l i s h e d w o r k o f P r i m a k o f f a n d S c h e d l ( r e f e r r e d t o e a r l i e r ) s u g g e s t s t h a t E. c o l i t R N A s may a l s o be t r a n s c r i b e d i n u n i t s o f t h r e e t o f i v e g e n e s . The a - g r o u p (38) c o u l d c e r t a i n l y be t r a n s c r i p t s o f t a n d e m g e n e s . R e c e n t w o r k by G h y s e n a n d C e l i s (40) s u g g e s t s t h a t t h e two t a ndem g e n e s f o r t R N A ^ y r o f 0 80 p s u p 3 ( K y o t o ) a r e t r a n s c r i b e d a s a s i n g l e p r e c u r s o r i n v i v o . P h age T4 c o d e s f o r a t l e a s t e i g h t t RNAs ( 4 1 ) . T h r e e p r e -c u r s o r t RNAs c a n be o b t a i n e d by a c r y l a m i d e g e l e l e c t r o p h o r e s i s a n d e a c h h a s b e e n shown t o c o n t a i n two t R N A s . I n one i n s t a n c e i t a p p e a r s t h a t a b a n d c o n a i n i n g two t R N A s i s n o t t h e p r i m a r y t r a n s c r i p t i o n p r o d u c t ( 4 2 ) . S i n c e i t i s known t h a t a l l e i g h t tRNA g e n e s a r e c l o s e l y c l u s t e r e d (43-45) i t i s p o s t u l a t e d t h a t a l l e i g h t a r e t r a n s c r i b e d a s a s i n g l e t r a n s c r i p t . Some w o r k h a s b e e n done w i t h h i g h e r o r g a n i s m s . Mammalian p r e c u r s o r tRNA i s known t o be s l i g h t l y l a r g e r t h a n m a t u r e tRNA 5. (see Burden review (18)). Its conversion to 4S RNA by crude mammalian extracts has been demonstrated. Kinetic studies con-firm a precursor product r e l a t i o n s h i p . Similar r e s u l t s have been obtained for insect tRNA (46,47,303). The nature of the RNA polymerase complex involved i n tRNA tr a n s c r i p t i o n i n E. c o l i has been the subject of considerable i n t e r e s t . In addition to the core enzyme (24) several other protein factors appear to be involved. In v i t r o studies on tRNA t r a n s c r i p t i o n suggest that sigma factor (48) i s necessary for correct i n i t i a t i o n of t r a n s c r i p t i o n and that rho factor (49) i s necessary for correct termination. In t h i s regard tRNA tr a n s c r i p t i o n i s l i k e mRNA tr a n s c r i p t i o n . An additional factor, p s i (50), has been implicated i n tRNA and rRNA synthesis. P s i factor has been shown to stimulate stable RNA synthesis up to 100 f o l d . An i n t e r e s t i n g observation, the implications of which w i l l be discussed l a t e r , i s that p s i factor i s i n f a c t the elongation factor complex TuTs (51). Multiple RNA polymerase a c t i v i t i e s with d i f f e r e n t template s p e c i f i c i t i e s have been observed i n crude E. c o l i extracts (52). A protein complex sedimenting at approximately 16S i s most active i n the synthesis of stable RNA. The molecular weight of t h i s complex i s consis-tent with the presence of TuTs and an a f f i n i t y for Tu has been demonstrated. In other work (53) i t has been shown that DNA must be activated before the e f f i c i e n t t r a n s c r i p t i o n of stable RNA can begin. This a c t i v a t i o n i s thought to involve a change i n the structure of the promoter region. I t has been suggested that p s i factor interacts with RNA polymerase so as to allow 6. the polymerase complex to recognize the activated promoter (54). Recent unpublished data (55) suggest that the protein H, a DNA unwinding protein, i s involved i n stable RNA promoter a c t i v a t i o n . Results Q.f Haseltine (56) and others have tended to argue against a role for p s i i n stable RNA synthesis. They observe extensive synthesis of stable RNA without the addition of p s i and l i t t l e or no stimulation when p s i i s added. These re s u l t s can be reconciled with those of Travers et aJL. i n that Haseltine worked with a system which was already maximally derepressed for stable RNA synthesis. The DNA had been disrupted so that stable RNA synthesis was no longer under natural control. Any RNA polymerase complex and not just the stable RNA polymerase complex (52) was active i n stable RNA t r a n s c r i p t i o n . The 3'0H Ends of tRNA The 3'0H ends of a l l functional tRNA molecules terminate in the t r i n u c l e o t i d e sequence pCpCpA (14). The absence of the terminal adenylate residue renders the tRNA incapable of accept-ing i t s cognate amino acid i n aminoacylation reactions. The presence or absence of one or more of the terminal nucleotides constitutes a p o t e n t i a l control mechanism to regulate the functional tRNA le v e l s of the cell.„ Although i n one instance i t has been suggested that the pCpCpA end i s a primary trans-c r i p t i o n product (35,36), there/is strong evidence that i t i s not i n several other instances (14,42,306). The enzymes respon-s i b l e for the addition of the three terminal nucleotides are c a l l e d tRNA nucleotidyltransferases. They are primarily bio-synthetic enzymes and have l i t t l e or no nuclease a c t i v i t y 7. i n v i v o ( 5 7 ) . O t h e r enzymes a r e i m p l i c a t e d i n t h e r e m o v a l o f t h e t e r m i n a l s e q u e n c e (58,59) s i n c e i t h a s b e e n d e m o n s t r a t e d t h a t t h e 3'0H t e r m i n a l n u c l e o t i d e s u n d e r g o r a p i d e x c h a n g e . I n n o r m a l l y g r o w i n g E. c o l i (60-62) a n d y e a s t (60) v i r t u a l l y a l l t RNA m o l e c u l e s a r e i n t a c t w i t h r e g a r d s t o t h e pCpCpA s e q u e n c e . I n n o n - l a c t a t i n g mammary g l a n d (63) s l o w g r o w i n g y e a s t ( 6 0 ) , B a c i l l u s s u b t i l i s s p o r e s (64,65) a n d u n f e r t i l i z e d s e a u r c h i n e g g s (66) t h e tRNAs a r e , t o a c o n s i d e r a b l e d e g r e e , m i s s i n g t h i s s e q u e n c e . S p o r u l a t i o n (64) a n d s e a u r c h i n egg f e r t i l i z a t i o n (67,68) r e s u l t i n i m m e d i a t e r e p a i r o f i n c o m p l e t e t e r m i n i . I n t h e c a s e o f B a c i l l u s s u b t i l i s s p o r u l a t i o n i t h a s b e e n c o n c l u s -i v e l y p r o v e n t h a t t h e a d d i t i o n o f t h e t e r m i n a l pCpCpA s e q u e n c e i s i n no manner r a t e l i m i t i n g i n o v e r a l l p r o t e i n s y n t h e s i s ( 6 4 ) . I n E. c o l i i t h a s b e e n shown (69) t h a t t h e amount o f n u c l e o t i d y l -t r a n s f e r a s e t h a t i s p r e s e n t i n t h e c e l l i s i n v a s t e x c e s s o f t h e amount n e e d e d f o r n o r m a l s y n t h e s i s a n d r e p a i r . Though n o t r a t e l i m i t i n g , 3*OH t e r m i n i r e p a i r i s r e l a t e d t o p r o t e i n s y n -t h e s i s i n t h a t t u r n o v e r o f t h i s s e q u e n c e i n E. c o l i i n c r e a s e s w i t h i n c r e a s e d g r o w t h r a t e (62) a n d v i r t u a l l y c e a s e s i n a m ino a c i d s t a r v e d E. c o l i o r c h l o r a m p h e n i c o l t r e a t e d E. c o l i ( 6 1 ) . A l t h o u g h t h e o r i e s h a v e b e e n p r o p o s e d (70) t h e r e i s a t t h i s t i m e no e v i d e n c e f o r a c e l l d i r e c t e d c o n t r o l m e c h a n i s m t o l i m i t o r a l t e r s p e c i f i c a l l y p r o t e i n s y n t h e s i s v i a t h e r e a d d i t i o n o r r e m o v a l o f pCpCpA. P o t e n t i a l f o r s u c h c o n t r o l d o e s h o w e v e r e x i s t . 8. The S t r i n g e n t R e s p o n s e I n b a c t e r i a t h e r e i s a s t r o n g c o r r e l a t i o n b e t w e e n n u c l e i c a c i d s y n t h e s i s a n d p r o t e i n s y n t h e s i s . E a r l y w o r k (71) showed t h a t when b a c t e r i a a r e d e p r i v e d o f a n e s s e n t i a l a m i n o a c i d , p r o t e i n s y n t h e s i s v i r t u a l l y c e a s e s and a d r a s t i c d e c r e a s e i n RNA s y n t h e s i s i s o b s e r v e d s h o w i n g t h a t RNA s y n t h e s i s i s c o u p l e d t o p r o t e i n s y n t h e s i s . M u t a t i o n s w e r e f o u n d a t a s i n g l e l o c u s , t h e r e l l o c u s , w h i c h r e l i e v e d t h e d e p e n d e n c y o f RNA s y n t h e s i s o n p r o t e i n s y n t h e s i s . The w i l d t y p e i s s a i d t o e x h i b i t a s t r i n g e n t r e s p o n s e t o amino a c i d d e p r i v a t i o n a n d t h e m u t a n t i s c o n s i d e r e d r e l a x e d w i t h r e g a r d t o t h i s c o n t r o l m e c h a n i s m . S t r i n g e n c y a c t s a s a n o v e r r i d i n g c o n t r o l m e c h a n i s m w h e n e v e r t h e r e i s a d e f i c i e n c y o f a m i n o a c y l - t R N A f o r a n y c o d o n i n t h e p r o c e s s o f b e i n g t r a n s l a t e d . When RNA s y n t h e s i s i s r e s t r i c t e d , i t i s t h e s t a b l e RNAs (tRNA a n d rRNA) w h i c h a r e m o s t d r a m a t i c a l l y a f f e c t e d . F u n c t i o n a l mRNA c o n t i n u e s t o be made f o r r e p r e s s i b l e , i n d u c i b l e a n d c o n s t i t u t i v e p r o t e i n s . C a s h e l a n d G a l l a n t (72,86) n o t e d t h a t s t r i n g e n t b u t n o t r e l a x e d c e l l s when d e p r i v e d o f a n e s s e n t i a l a m i n o a c i d a c c u m u l a t e two u n u s u a l n u c l e o t i d e s MSI a n d M S I I . MSI a n d M S I I w e r e s u b s e q u e n t l y i d e n t i f i e d a s ppGpp an d pppGpp r e s p e c t i v e l y ( 3 0 7 ) . I t h a d b e e n e a r l i e r shown t h a t a n t i b i o t i c s w h i c h f u n c t i o n a s r i b o s o m e i n h i b i t o r s p r o d u c e d t h e r e l a x e d p h e n o t y p e i n g e n e t i c a l l y s t r i n g e n t c e l l s . T h e s e 1 I t i s i m p o r t a n t t o t h i s t h e s i s t h a t t h i s phenomenon be d i s c u s s e d a t some l e n g t h b u t b e c a u s e o f t h e enormous v o l u m e o f l i t e r a t u r e o n t h e s u b j e c t , a minimum number o f o r i g i n a l r e f e r e n c e s a r e u s e d . The r e a d e r c a n f i n d m o s t o f t h e o r i g i n a l r e f e r e n c e s i n a c o m p r e -h e n s i v e r e v i e w a r t i c l e b y Ryan a n d B o r e k ( 1 9 ) . same a n t i b i o t i c s likewise prevent MSI and MSII accumulation. Cashel and Gallant proposed that the MS nucleotides were syn-thesized i n an i d l i n g reaction i n protein synthesis and that the MS nucleotides must i n some manner p r e f e r e n t i a l l y l i m i t stable RNA synthesis. Studies by several workers established that the stringent response was not li m i t e d to amino acid dep-r i v a t i o n but that the MS nucleotides were i n fact part of a control mechanism that i s operative during stepdown t r a n s i t i o n (73-75) and NaCl i n h i b i t i o n (76) etc. (77-79) i . e . a general control mechanism for the synthesis of stable RNA. Relaxed mutants accumulate far less MS nucleotide when starved for amino acids but respond to stepdown t r a n s i t i o n i n b a s i c a l l y the same manner as do stringent c e l l s . In a series of elegant experiments i n v i t r o Haseltine and co-workers (80) showed that (a) MS nucleotides are synthesized on the ribosome; (b) t h i s synthesis requires the G translocation factor and a protein factor which can be washed o f f ribosomes by high s a l t concentration; (c) synthesis of MS nucleotides i s suppressed by i n h i b i t o r s of G factor; (d) GDP i s the precursor for MSI and GTP for MSII with ATP supplying the additional 3'OH phosphates; (e) the high s a l t wash factor appears functionally absent from the ribosomes of genetically relaxed c e l l s . Since a l l r e l are leaky (81) mutants some of t h i s protein i s present. This may explain why r e l syn-thesize MS nucleotides during stepdown t r a n s i t i o n . Perhaps the protein i s functional with respect to step down t r a n s i t i o n but not with respect to amino acid deprivation. Non-ribosomal syn-thesis of MS nucleotides i n v i t r o has recently been achieved (82) 10. Kinetic data suggest that the MS nucleotides bring about the p r e f e r e n t i a l shut o f f of stable RNA synthesis. Although MS nucleotides are potent i n h i b i t o r s of many c e l l processes i t i s very important to determine pre c i s e l y how they can d i f f e r -entiate between mRNA tr a n s c r i p t i o n and stable RNA tr a n s c r i p t i o n so as to i n h i b i t one but not the other. This d i s t i n c t i o n i s possible because stable RNA tr a n s c r i p t i o n uniquely requires the protein factor p s i . P s i factor has been shown to be strongly i n h i b i t e d by ppGpp (50). The 16S RNA polymerase complex referred to e a r l i e r i s likewise strongly i n h i b i t e d while RNA polymerase complexes not implicated i n stable RNA synthesis are not (52). Since p s i i s i n fac t the elongation complex TuTs as previously mentioned the d i r e c t coupling of protein synthesis and stable RNA synthesis i s explained. Some controversy has arisen i n the l i t e r a t u r e (83-85) because ppGpp was not i n h i b i t o r y of stable RNA t r a n s c r i p t i o n i n v i t r o . Under the experimental conditions used, the DNA i s transcribed i n a nonphysiological manner i n that the authors attempted to make as much t r a n s c r i p t as possible and i n doing so they used DNA which was i n a conformation where a l l natural control i s l o s t . The a b i l i t y of the c e l l to regulate the synthesis of s p e c i f i c RNAs v i a the heterogeneity of the RNA polymerase complexes becomes meaningless i n that a l l poly-merase complexes recognize a l l promoters because the DNA i s i n an unnatural state. A f i n a l point to be mentioned i s that the r e l gene function should be considered an overriding control mechanism. Other 1 1 . possible control mechanisms for tRNA biosynthesis could for sur v i v a l reasons be overridden by i t . tRNA synthesis i s not simply related to gene dosage but must involve highly s p e c i f i c cytoplasmic control (87,88). S p e c i f i c Control of tRNA Synthesis The previous discussion shows that control of tRNA biosyn-thesis on an o v e r a l l l e v e l has been well established. Because of the central role of tRNA i n protein synthesis i t i s important to determine i f i n d i v i d u a l tRNAs are subject to s p e c i f i c control mechanisms. Possibly a l l tRNAs are made i n quantities dictated by the needs of the c e l l . A l t e r n a t i v e l y , some tRNAs may be con-t r o l l e d i n a passive, e s s e n t i a l l y c o n s t i t u t i v e manner, while the synthesis of selected key tRNAs i s c a r e f u l l y regulated. Lastly i t i s possible that a l l tRNAs are synthesized i n a con s t i t u t i v e manner as a function of polymerase lev e l s etc. In many studies done to date the acceptance of tRNA for a pa r t i c u l a r amino acid has been measured i n order to correlate any changes i n le v e l s observed with the state of the system. As there are usually several tRNA isoacceptors for any p a r t i c u l a r amino acid most studies have included a chromatographic analysis of the r e l a t i v e amounts or d i s t r i b u t i o n of i n d i v i d u a l i s o -acceptors. Often new chromatographic peaks are observed. I t should be remembered that such peaks are of several possible o r i g i n s . They may r e f l e c t the tr a n s c r i p t i o n of tRNA genes not previously transcribed. They may represent undermodified forms of the normally observed isoacceptor and l a s t l y they may rep-resent normal isoacceptors i n an altered physical state. The most thorough studies attempt to d i f f e r e n t i a t e between these p o s s i b i l i t i e s . In order to attempt an understanding of any possible control mechanism for tRNA synthesis i t i s necessary to know the i d e n t i t y and hence o r i g i n of a l l the i n d i v i d u a l tRNA isoacceptors. Functional Adaptation A term that Garel (89) has coined which represents one form of tRNA control i s "functional adaptation". I f a c e l l i s able to regulate the amount of tRNA f o r a p a r t i c u l a r amino acid, i t i s to the advantage of the c e l l to synthesize tRNAs i n propor-t i o n to the amino acid composition of the proteins that i t i s making at a p a r t i c u l a r time. This concept has been tested i n several systems. The s i l k gland of Bombyx mori has a posterior part, the secreteur, which becomes sp e c i a l i z e d for the synthesis of the protein f i b r o i n , and a middle portion, the reservoir, which be-comes speci a l i z e d for the synthesis of the protein s e r i c i n . During development there i s a ten f o l d increase i n the tRNA of the secreteur and a f i v e f o l d tRNA increase i n the reservoir. The synthesis of tRNA i s such that those tRNAs capable of accepting the four amino acids predominant i n f i b r o i n (glycine, alanine, serine and tyrosine) constitute 2/3 of the t o t a l tRNA population at the 8th day of development (90,91). A l i n e a r c o r r e l a t i o n exists between these predominant amino acids of f i b r o i n and the corresponding tRNAs during the secretion phase (92). The work of Suzuki and Brown (93) on the oligonucleotide d i s t r i b u t i o n of the i s o l a t e d f i b r o i n mRNA suggests that the messenger RNA mainly contains the codon GCU for alanine, the codons GGA and GGU for glycine and the codon UCA for serine. Based upon the coding responses of the fractionated isoacceptors for alanine and glycine, Garel et a l . (94) and Chen and Siddiqui (95) suggest that the isoacceptor d i s t r i b u t i o n for these amino acids i s q u a l i t a t i v e l y adapted to t h e i r t r a n s l a t i o n a l function for f i b r o i n mRNA. In the reservoir, tRNAs for serine, glycine, aspartate, glutamate and alanine, the f i v e major amino acids of s e r i c i n , likewise constitute 2/3 of the post ribosomal super-natant tRNA pool by the 7th day. While that group of tRNAs most important to both f i b r o i n and s e r i c i n synthesis reach peak concentrations at 7 to 8 days a second group of tRNAs can be distinguished. This second group reaches maximum pool concen-trations at 4 to 5 days where upon which t h e i r synthesis appar-ently ceases. These tRNAs would appear to be necessary for the protein synthesis which precedes f i b r o i n and s e r i c i n production. A mechanism therefore appears to select for the continued syn-thesis of cer t a i n tRNAs and the decreased synthesis of others. The significance of such results i s that they show that a c e l l i s not g e n e t i c a l l y programmed to have a s p e c i f i c nonvariable tRNA population. tRNA populations can change i n a highly s p e c i f i c manner. Functional adaptation has been suggested i n several other systems. I t has been reported (96-98,302) that tRNA of rabbit reticulocytes i s specia l i z e d for hemoglobin synthesis. Garel et a l . (99) compared the tRNA from the r e l a t i v e l y s p e c i a l i z e d i n t e r n a l c o r t i c a l zone c e l l s of bovine lens tissue with those from the less s p e c i a l i z e d external c o r t i c a l zone. They observed that for 10 of 12 tRNA species examined, the l e v e l of tRNA was functionally adapted for the c r y s t a l l i n synthesis c h a r a c t e r i s t i c of the i n t e r n a l c o r t i c a l zone. Ortwerth (100) noted major d i f f e r -ences i n amino acid acceptance l e v e l s and tRNA isoacceptor d i s -t r i b u t i o n on RPC-2 between mammalian lens and muscle tissue. He postulated tissue s p e c i a l i z a t i o n of tRNA populations as a function of the proteins being synthesized. Yang (101) reported a c o r r e l a t i o n of seven representative tRNAs with the amino acid composition for IgF myeloma proteins i n MOPC31C plasma c e l l tumor. Lactating mammary gland i s special i z e d for the synthesis of casein, a glutamate-rich glycine-poor protein. Elska et. a l . (102) observed that at la c t a t i o n there i s a 78% increase i n glutamate acceptance and a 62% decrease i n glycine acceptance compared to v i r g i n a l mammary tRNA. They also noted markedly increased i n vivo aminoacylation l e v e l s at the onset of l a c t a t i o n . Rat granulation tissue i s spec i a l i z e d for the synthesis of large amounts of pr o l i n e - and gl y c i n e - r i c h collagen. As the granulated tissue developed, 30% increases i n glycine and proline acceptances were observed (103). Chromatographically one of three glycine isoacceptors was shown to undergo a major increase i n size r e l a t i v e to the two others. Estrogen-induced synthesis of s e r i n e - r i c h phosvitin i n rooster l i v e r s r e s u l t s i n a 25% increase i n t o t a l serine acceptance (104) due to an i n c r e a s e i n the content o f the minor Ser Ser i s o a c c e p t o r tRNA^^ and the major i s o a c c e p t o r t R N A ^ j (105-107) . These changes were observed t o be r e v e r s e d upon the t e r m i n a t i o n of p h o s v i t i n s y n t h e s i s ( 1 0 5 ) . In systems where a wide v a r i e t y of p r o t e i n s i s made a t p o s s i b l y v e r y d i f f e r e n t r a t e s a c o n t r o l mechanism such as f u n c t i o n a l a d a p t a t i o n i s more d i f f i c u l t t o r e c o g n i z e . However, Yamane ( 1 0 8 ) , s t u d y i n g f o u r d i f f e r e n t b a c t e r i a , observed t h a t the l e v e l s o f f i v e aminoacyl-tRNA roughly c o r r e l a t e d w i t h the amino a c i d composition o f t o t a l p r o t e i n f o r each bacterium. Since i t has been shown t h a t the tRNAs of growing b a c t e r i a are n e a r l y completely aminoacylated (109,110) t h i s i s e q u i v a l e n t to say i n g t h a t the tRNA p o p u l a t i o n o f b a c t e r i a i s f u n c t i o n a l l y adapted t o the amino a c i d composition of the bulk o f the p r o t e i n being s y n t h e s i z e d . tRNA During D i f f e r e n t i a t i o n and Development D i f f e r e n t i a t i o n and development are p o t e n t i a l l y tRNA d i r e c -t e d and c o n t r o l l e d p r o c e s s e s . By r e g u l a t i o n of the s i z e and nature o f the tRNA p o o l , c e l l s and t i s s u e s c o u l d a l t e r the r a t e of t r a n s l a t i o n o f c e r t a i n mRNAs or even terminate t h a t t r a n s l a -t i o n . Numerous s t u d i e s have been done on these t o p i c s and c e r t a i n l y i n a few i n s t a n c e s a l t e r e d t r a n s l a t i o n a l a b i l i t y or e f f i c i e n c y i s i m p l i c a t e d . The s y n t h e s i s and degree of m o d i f i c a -t i o n o f s p e c i f i c tRNAs i s the instrument which the c e l l uses to achieve these ends. In p l a n t s , comparing e l e v e n tRNAs of wheat embryos and s e e d l i n g s , V o i d and Sypherd (111) found minor d i f f e r e n c e s f o r acceptors of l y s i n e , proline and serine. Also working with Phe wheat Shugart (112) noted chromatographic differences i n tRNA obtained from growing and nongrowing tissue (apical end vs. basal end of the same l e a f ) . Minimal differences i n chromato-graphic patterns (1135) of cytoplasmic tRNAs have been found for four d i f f e r e n t developmental stages during cotyledon embryogene-s i s and germination i n cotton. Soybean cotyledons during sen-escense show a three f o l d increase i n tRNAr „ -, c (114) 5 and 6 r e l a t i v e to the other leucine isoacceptors. As t o t a l leucine acceptance decreases markedly i t would appear that the synthesis of tRNA^_^ i s p r e f e r e n t i a l l y r e s t r i c t e d while that of the 5th and 6th isoacceptors i s not. In animals t o t a l tRNA acceptance was shown to decrease with age i n mouse brain (115). Rat brain likewise showed an o v e r a l l decrease i n tRNA with age. However, ce r t a i n tRNAs were enriched r e l a t i v e to others (116). Quantitative but no q u a l i t a t i v e changes were observed i n aging rat spleen (117). Mouse placental tRNA was shown to double the number of peaks coding f o r h i s t i -dine at the 14th day of pregnancy (118). Different methionine and arginine tRNA p r o f i l e s were ob-tained for tRNA from l a r v a l b u l l frog (Rana catesbeiana) erythrocytes and adult erythrocytes (119). Minor differences were noted between Xenopus embryo and adult with respect to codon response for isoleucine, glutamate and arginine (120). Comparing the encysted gastrula and nauplius l a r v a l stages of brine shrimp, Bagshaw et a l . (121) showed chromatographically quantitative differences i n acceptors for nine amino acids and 17. no d i f f e r e n c e s a t a l l f o r n i n e o t h e r s . C o m p a r i n g f i v e s t a g e s o f s e a u r c h i n d e v e l o p m e n t w i t h r e s p e c t t o f o u r d i f f e r e n t amino a c i d s , s m a l l c h a n g e s w e r e o b s e r v e d i n tRNA * a n d a new tRNA was o b s e r v e d a t g a s t r u l a t i o n ( 1 2 2 ) . An e x t r a s e r i n e i s o a c c e p t o r i s s e e n i n t r o p h o z o i t e s o f A c o n t h a m o e b a c a s t e l l a n i i i n a d d i t i o n t o t h e two f o u n d i n t h e p r e c y s t ( 1 2 3 ) . I t i s n o t s u f f i c i e n t s i m p l y t o o b s e r v e c h a n g e s i n tRNA p o p u l a t i o n s . I t i s n e c e s s a r y t o d e t e r m i n e how t h e s y s t e m b r o u g h t s u c h tRNA c h a n g e s a b o u t . R e c e n t w o r k b y W h i t e e t a l . (124,125) o n t h e d e v e l o p m e n t o f D r o s o p h i l a d o e s t h a t . W h i l e a g r e a t many q u a n t i t a t i v e c h r o m a t o g r a p h i c d i f f e r e n c e s w e r e o b s e r v e d b e t w e e n 1 s t i n s t a r , 3 r d i n s t a r a n d a d u l t , a d e f i n i t e p a t t e r n e m e r g e d f o r t h o s e tRNAs w h i c h a c c e p t h i s t i d i n e , t y r o s i n e , a s p a r t a t e a n d a s p a r a g i n e . E a c h g r o u p o f i s o a c c e p t o r s c o u l d be d i v i d e d i n t o 6 a n d y f o r m s . T h e s e f o r m s w e r e shown i n two i n s t a n c e s t o be h o m o g e n e i c i . e . d e r i v e d f r o m t h e same gene b u t d i f f e r i n g i n t h e d e g r e e o f m o d i f i c a t i o n o f m i n o r n u c l e o s i d e s ; i n t h i s i n s t a n c e Q. D u r i n g d e v e l o p m e n t , t h e t o t a l a c c e p t a n c e o f t h e s e f o u r a m ino a c i d s b y tRNA r e m a i n s r e l a t i v e l y c o n s t a n t b u t t h e amounts o f t h e 6 f o r m s d e c r e a s e f r o m t h e egg t o t h e t h i r d i n s t a r and t h e r e a f t e r i n c r e a s e u n t i l two weeks a f t e r e c l o s i o n . The amounts o f t h e y f o r m s v a r y i n v e r s e l y . The 6 f o r m c o n t a i n s Q. S i n c e t h e p r e s e n c e o f Q c h a n g e s t h e c o d i n g c h a r a c t e r i s t i c s o f t h e tRNA t h i s t y p e o f b a s e m o d i f i c a t i o n i s p o t e n t i a l l y a n i m p o r t a n t t r a n s l a t i o n a l c o n t r o l m e c h a n i s m . Work b y I l a n (126,127) h a s s u g g e s t e d t h a t i n T e n e b r i o  m o l i t o r a new l e u c i n e a c c e p t o r a c t i v i t y i s f o u n d i n t h e 7 t h 18. day pupae but not i n 1st day or juvenile hormone-treated pupae. The mRNA for adult c u t i c l e i s present at an e a r l i e r time but i s not translated u n t i l the appearance of t h i s new tRNA L e u. The implication i s that a new tRNA i s required to translate a l i m i t i n g leucine codon i n the adult c u t i c l e mRNA. Hormonal. E f f e c t s on tRNA Hormone-mediated changes i n tRNA have been observed i n several systems. Estrogen induced phosvitin synthesis and the Ser accompanying changes i n the tRNA p r o f i l e (104) have already been discussed i n terms of tRNA functional adaptation. Possible changes i n tRNA i n the meal worm Tenebrio molitor (126) associated with juvenile hormone have been discussed i n terms of development and d i f f e r e n t i a t i o n . There are many other i n -stances where hormones influence tRNA populations but where the relati o n s h i p i s less obvious. Busby and Hele (128) observed estrogen-induced changes i n the tRNA L^ s of chicken l i v e r . The f i r s t isoacceptor o f f a methylated albumin kieselgur. column, tRNA^ s, was substa n t i a l l y increased i n amount r e l a t i v e to tRNA^ s. Several changes i n t o t a l amino acid acceptance by tRNA for other amino acids were also noted. A new tRNA species has been detected during d i f f e r e n t i a t i o n of bovine mammary gland (129) and as noted e a r l i e r (102) large changes i n amino acid acceptance are ob-served during l a c t a t i o n . Changes i n tRNA have likewise been observed during b u l l frog tadpole metamorphosis induced by triiodothyronine (13 0). A l t m a n and c o - w o r k e r s (131) n o t e d t h a t a d m i n i s t r a t i o n o f h y d r o c o r t i s o n e t o f e m a l e r a t s g a v e r i s e t o a new tRNA w h i c h was t r a n s i e n t a n d s u b s e q u e n t l y d i s a p p e a r e d . R e m o v i n g hormone s e c r e t i n g o r g a n s o f t e n a f f e c t s t h e tRNA p o p u l a t i o n o f t a r g e t t i s s u e . T h y r o i d e c t o m y g a v e r i s e t o a l t e r e d p r o f i l e s f o r r a t l i v e r t R N A L y s a n d t R N A P h e ( 1 3 2 ) . O v a r i e c t o m y S e r c h a n g e d t h e tRNA p r o f i l e o f p i g u t e r u s b u t n o t p i g l i v e r ( 1 3 3 ) . H y p o p h y s e c t o m i z e d r a t s show up t o 30% d e c r e a s e s i n s p e c i f i c a m i no a c i d a c c e p t o r a c t i v i t i e s (134) a l t h o u g h o n l y m i n o r c h r o m a t o g r a p h i c d i f f e r e n c e s w e r e o b s e r v e d . When g r o w t h hormone was a d m i n i s t e r e d t o h y p o p h y s e c t o m i z e d r a t s , i n c r e a s e s i n a m i no a c i d a c c e p t a n c e s w e r e o b s e r v e d (135) a n d two new t R N A A s ^ i s o a c c e p t o r s w e r e d e t e c t e d (13 6 ) . T h e s e new i s o a c c e p -t o r s w e r e shown,however, t o be c h a r a c t e r i s t i c o f g r o w i n g l i v e r a n d n o t a s p e c i f i c h o r m o n a l r e s p o n s e . O r g a n a n d T i s s u e tRNA D i f f e r e n c e s Q u a n t i t a t i v e a n d q u a l i t a t i v e d i f f e r e n c e s i n tRNA p o p u l a t i o n s o f d i f f e r e n t o r g a n s , t i s s u e s a n d o r g a n e l l e s h a v e o f t e n b e e n o b s e r v e d . I t i s t e m p t i n g t o s p e c u l a t e t h a t s u c h d i f f e r e n c e s r e p r e s e n t f u n c t i o n a l a d a p t a t i o n i n some manner t o t h e p r o t e i n s b e i n g s y n t h e s i z e d i n e a c h i n s t a n c e . T h e s e d i f f e r e n c e s , u n l e s s a l r e a d y p r e v i o u s l y d i s c u s s e d , w i l l n o t be e l a b o r a t e d u p o n any f u r t h e r h e r e . T h i s t h e s i s w i l l a t t e m p t t o c o n c e n t r a t e o n t h o s e tRNA c o n t r o l m e c h a n i s m s a n d tRNA d i f f e r e n c e s w h i c h a r e b e s t u n d e r s t o o d a n d o n t h o s e w h i c h a r e m o s t r e l a t e d t o t h e e x p e r i m e n t a l w o r k t o be d e s c r i b e d . E a r l i e r r e f e r e n c e s a r e c o n t a i n e d i n a r e v i e w b y S u e o k a a n d Kano S u e o k a ( 1 5 ) . The r e v i e w b y L i t t a u e r a n d I n o u y e (137) i s a l s o h e l p f u l . A d d i t i o n a l r e f e r e n c e s t o tRNA c o m p o s i t i o n o f o r g a n e l l e s c a n be o b t a i n e d f r o m t h e p a p e r by R i t t e r a n d B u s c h ( 1 3 8 ) . tRNA a n d Phage I n f e c t i o n P h age i n f e c t i o n o f E. c o l i h a s b e e n shown t o b r i n g a b o u t c h a n g e s i n t h e o b s e r v e d tRNA p o p u l a t i o n . P hage T5 i s known t o h a v e a s a p a r t o f i t s genome a t l e a s t 14 tRNA g e n e s (139) a n d phage T4 i s t h o u g h t t o c o d e f o r a t l e a s t 8 tR N A s ( 4 1 ) . The T4 tRNA g e n e s a r e t r a n s c r i b e d f r o m t h e l i g h t s t r a n d o f T4 DNA (44) an d a r e c l o s e l y c l u s t e r e d ( 1 4 0 ) . T - e ven p h a g e s a r e u n i q u e i n t h a t t h e y c o d e f o r a h i g h l y s p e c i f i c n u c l e a s e w h i c h i n a c t i v a t e s tRNA^ o f t h e h o s t ( 1 4 1 -14 5 ) . A p p r o x i m a t e l y 60% o f t h e h o s t t R N A ^ e u i s r e n d e r e d i n a c -t i v e b y a s i n g l e c l e a v a g e w h i c h g i v e s two s p e c i f i c f r a g m e n t s ; o n e , 48 n u c l e o t i d e s l o n g f r o m t h e 5'-end a n d t h e o t h e r 39 n u c l e o t i d e s l o n g a r i s i n g f r o m t h e 3'-end ( 1 4 6 ) . t R N A ^ e u r e c o g n i z e s CUG, a t r i p l e t w h i c h i s r a r e i n T - e v e n phage mRNA ( 1 4 1 ) . I t h a s t h e r e f o r e b e e n p o s t u l a t e d t h a t t h e i n a c t i v a t i o n o f t R N A 1 l i m i t s t h e a b i l i t y o f t h e h o s t c e l l t o t r a n s l a t e i t s own mRNA a n d i s one o f t h e m e c h a n i s m s f o r s h u t t i n g o f f h o s t p r o t e i n s y n t h e s i s ( 1 4 1 , 1 4 7 ) . An i n t e r e s t i n g e x p l a n a t i o n f o r t h e p r e s e n c e o f tRNA g e n e s i n t h e T4 genome h a s r e c e n t l y b e e n p u t f o r w a r d b y W i l s o n ( 4 5 ) . Numerous T4 s t r a i n s a r e a v a i l a b l e c a r r y i n g c h a r a c t e r i z e d d e l -e t i o n s i n t h e T4 tRNA g e n e s . W i l s o n (45) d e m o n s t r a t e d t h a t burst size of the tRNA d e f i c i e n t strains i s inversely propor-t i o n a l to the size of the deletion. No single tRNA was more important than any other and strains deleted i n a l l tRNA genes are s t i l l v iable but have a 40% reduction i n burst s i z e . Using SDS polyacrylamide gel electrophoresis he was able to correlate the amounts of two t a i l f i b e r proteins, P34 and P37, with the size of the tRNA deletion. I t would seem that phage proteins, p a r t i c u l a r l y P34 and P3 7, contain codons for which the host has a l i m i t i n g amount of tRNA and thus the T4 tRNAs ensure optimal rates of protein synthesis by supplementing the tRNA content of the host. Scherberg and Weiss (148) have shown i n t r i p l e t binding studies that the T4 a r g i n y l - , g l y c y l - , i s o l e u c y l - and l e u c y l - tRNAs recognize those codons for which the host E. c o l i has the l e a s t amount of tRNA. I t i s also possible that labora-tory E. c o l i s trains are not the natural host for T4 and that T4 tRNA i s absolutely required i n another host. There i s some experimental evidence for t h i s (45). Infection of E. c o l i by the RNA phage Qg i s also i n t e r e s t i n g i n that a f t e r i n f e c t i o n Pro tRNA responds to a poly C message two to three times less Pro e f f i c i e n t l y than the tRNA from the uninfected c e l l s (149). Considering the limited coding capacity of the Q3 genome i t i s hard to v i s u a l i z e how Q3 i n f e c t i o n brings about t h i s response tRNA and Cancer A great deal of study has been undertaken to characterize cancerous and p o t e n t i a l l y cancerous systems i n biochemical terms In such tissues major changes i n protein synthesis, both quan-t i t a t i v e and q u a l i t a t i v e , are usually observed. Since tRNA has b e e n p o s t u l a t e d a s a p o t e n t i a l m e d i a t o r o f t r a n s l a t i o n a l c o n t r o l , a b e r r a n t tRNA s y n t h e s i s i s p o t e n t i a l l y a c a u s a t i v e a g e n t o f c a n c e r ( 3 0 8 , 1 5 0 ) . Much work, h a s b e e n done t o t e s t t h i s t h e o r y c f . S u e o k a (15) a n d L i t t a u e r ( 1 3 7 ) . D e s p i t e a n enormous w e a l t h o f l i t e r a t u r e o n t h e s u b j e c t t h e e x a c t r e l a t i o n s h i p s b e t w e e n c a n c e r a n d tRNA a r e s t i l l p o o r l y d e f i n e d . M a j o r q u a n t i t a t i v e a n d q u a l i t a t i v e tRNA c h a n g e s h a v e b e e n o b s e r v e d i n some s y s t e m s ( 1 5 1 , 1 5 2 ) . O t h e r s y s t e m s show m i n i m a l tRNA d i f f e r e n c e s i n t h e n e o p l a s t i c s t a t e ( 1 5 3 , 1 5 4 ) . A l t e r e d tRNA m e t h y l a s e a c t i v i t y i s common an d p r o b a b l y a c c o u n t s f o r many o f t h e q u a l i t a t i v e d i f f e r -e n c e s o b s e r v e d . The t u r n i n g o n o r s h u t t i n g o f f o f s p e c i f i c t RNA g e n e s i s n e v e r t h e l e s s a c r e d i b l e p o s s i b i l i t y . The p o t e n t i a l f o r s u c h c o n t r o l m e c h a n i s m s i s d i s c u s s e d i n d e t a i l i n s e v e r a l o t h e r p a r t s o f t h i s t h e s i s . tRNA a n d G r o w t h C o n d i t i o n s The c o m p o s i t i o n o f tRNA o f l o w e r o r g a n i s m s i s d e p e n d e n t u pon t h e g r o w t h c o n d i t i o n o r g r o w t h s t a g e . D i f f e r e n c e s i n i s o a c c e p t o r Phe T r D d i s t r i b u t i o n f o r tRNA a n d tRNA ^ h a v e b e e n o b s e r v e d f o r Rhodopseudomonas s p h e r o i d e s g r o w n a e r o b i c a l l y a s c o m p a r e d t o a n a e r o b i c a l l y ( 1 5 5 ) . P r o t e i n s y n t h e s i s i s known t o d i f f e r i n t h e two s t a t e s . N e u r o s p o r a c r a s s a w h i c h was g rown w i t h o u t s h a k i n g h a d a t R N A A r g p o p u l a t i o n w h i c h r e a d i l y b o u n d t o p o l y (C,G,A) b u t n o t p o l y (A,G) i n r i b o s o m e b i n d i n g a s s a y s . H o w e v e r , s h a k e n c u l t u r e s h a d t R N A A r g w h i c h s t r o n g l y b o u n d p o l y (A,G) a n d b o u n d p o l y (C,G,A) t o a l e s s e r e x t e n t ( 1 5 6 ) . 23. tRNA p r o f i l e s o f B a c i l l u s h a v e b e e n shown t o c h a n g e u n d e r s e l e c t e d c i r c u m s t a n c e s . An a d d i t i o n a l t R N A L y s i s p r e s e n t i n v e g e t a t i v e o r s p o r u l a t i n g c e l l s w h i c h i s p r e s e n t i n v e r y l o w c o n c e n t r a t i o n s i n s p o r e s ( 1 5 7 , 1 5 8 ) . L a z z a r i n i . (159) p r o p o s e d t h a t t h e e x t r a p e a k was g r o w t h medium d e p e n d e n t . O t h e r s d i s -a g r e e ( 1 5 8 ) . C h a n g e s i n t R N A V a l d u r i n g s p o r u l a t i o n h a v e b e e n r e p o r t e d ( 1 6 1 - 1 6 3 ) . S i m i l a r l y d i f f e r e n t t y r o s i n e tRNAs p r e -d o m i n a t e i n e x p o n e n t i a l ( f o r m I ) a n d s t a t i o n a r y ( f o r m I I ) s t a t e s . A r e c e n t p a p e r b y V o i d (164) c o m p a r i n g tRNAs f r o m s p o r e s a n d e x p o n e n t i a l l y g r o w i n g c e l l s , b y t h e i m p r o v e d c h r o m a t o g r a p h i c t e c h n i q u e RPC-5, shows no tRNA d i f f e r e n c e s f o r tRNAs a c c e p t i n g p h e n y l a l a n i n e , v a l i n e , a l a n i n e , a s p a r t a t e , i s o l e u c i n e , p r o l i n e , m e t h i o n i n e a n d h i s t i d i n e ; c h a n g i n g r a t i o s f o r t R N A s a c c e p t i n g g l y c i n e , t y r o s i n e , l e u c i n e , s e r i n e , t h r e o n i n e a n d a s p a r a g i n e a n d q u a l i t a t i v e d i f f e r e n c e s f o r tRNAs a c c e p t i n g l y s i n e , g l u t a m a t e a n d t r y p t o p h a n . S k j o l d e t a l . (165) f o u n d t h a t i n E. c o l i t h e tRNA t o DNA r a t i o i n c r e a s e d s u b s t a n t i a l l y w i t h i n c r e a s i n g g r o w t h r a t e b u t t h e p e r c e n t o f t h e t o t a l a c c e p t a n c e o f i n d i v i d u a l amino a c i d s r e m a i n e d e s s e n t i a l l y c o n s t a n t . They o b s e r v e d no c h r o m a t o g r a p h i c d i f f e r e n c e s f o r tRNAs f o r s e v e n amino a c i d s when e x a m i n e d by B D - c e l l u l o s e c h r o m a t o g r a p h y . B a r t z e t a l . (166) r e p o r t no c h r o m a t o g r a p h i c d i f f e r e n c e s i n E. c o l i tRNA f r o m e a r l y , m i d Phe and l a t e l o g c e l l s . T h ey d i d o b s e r v e a new tRNA i s o a c c e p t o r i n s t a t i o n a r y c e l l s . M s 2 i 6 A a n d i 6 A c o n t e n t was shown t o be h i g h e s t i n e a r l y l o g c e l l s a n d much l o w e r i n l a t e r s t a g e s o f g r o w t h . Some d i f f e r e n c e s i n amino a c i d a c c e p t a n c e b e t w e e n v a r i o u s s t a g e s w e r e o b s e r v e d . A much l o w e r l e v e l o f t R N A ^ e u was o b s e r v e d i n s t a t i o n a r y c e l l s t h a n i n e x p o n e n t i a l l y g r o w i n g c e l l s (167). G r o s s a n d Raab (168) r e p o r t e d m a j o r d i f f e r e n c e s T v r i n tRNA J p r o f i l e s b e t w e e n e a r l y l o g a n d l a t e l o g E. c o l i c e l l s . I n e a r l y l o g t h e t w o i s o a c c e p t o r s a r e p r e s e n t i n a p p r o x -i m a t e l y e q u a l amounts b u t i n l a t e l o g t R N A ^ 1 " i s p r e d o m i n a n t . I t h a s b e e n r e p o r t e d (169) t h a t a n a e r o b i c a l l y g rown E. c o l i l i e h a v e a n a l t e r e d tRNA p r o f i l e . E. c o l i (170) grown o n l e s s — 7 Phe t h a n 10 M i r o n h a v e o ne o r two a d d i t i o n a l tRNA i s o a c c e p t o r s L i k e w i s e E. c o l i g r o w n o n l o w p h o s p h a t e m e d i a h a v e a n a d d i t i o n a l P h e tRNA , t h e amount o f w h i c h i s d e p e n d e n t upon t h e g r o w t h r a t e , s l o w e r g r o w i n g c e l l s h a v i n g more (171). B a c t e r i a h a v e b e e n g r o w n u n d e r a d v e r s e c o n d i t i o n s a n d t h e tRNAs c h a r a c t e r i z e d . S u c h g r o w t h c o n d i t i o n s d i s r u p t t h e n o r m a l s y n t h e s i s a n d m a t u r a t i o n o f tRNA. E a r l y w o r k b y W a t e r s (172) showed t h a t c h l o r a m p h e n i c o l t r e a t e d E. c o l i h a d a l t e r e d c h r o m a t o g r a p h i c p r o f i l e s f o r tRNA t R N A P h e a n d t R N A T y r . W a t e r s e t . a l . (173) c o n f i r m e d t h e e a r l y r e s u l t s a n d showed t h e f o r m a t i o n o f new p e a k s t o b e a g e n e r a l phenomenon. I n a d d i t i o n , t h e y showed t h a t tRNA made d u r i n g c h l o r a m p h e n i c o l t r e a t m e n t d i f f e r s f r o m n o r m a l tRNA i n t h a t i t h a s 60 - 70% l e s s o f t h e m i n o r n u c l e o t i d e s 4 - t h i o u r i d i n e a n d d i h y d r o u r i d i n e . M o s t m e t h y l a t e d m i n o r n u c l e o t i d e s w e r e e s s e n -t i a l l y n o r m a l . The tRNA s y n t h e s i z e d d u r i n g c h l o r a m p h e n i c o l t r e a t m e n t was a b l e t o f u n c t i o n n o r m a l l y i n a n i n v i t r o hemo-g l o b i n s y n t h e s i z i n g s y s t e m . The d i h y d r o u r i d i n e a n d 4 - t h i o u r i -d i n e c h a n g e s h a v e b e e n i n d e p e n d e n t l y d e m o n s t r a t e d b y o t h e r w o r k e r s (174). A l l d a t a a r e c o n s i s t e n t w i t h t h e new tRNA i s o -a c c e p t o r s b e i n g u n d e r m o d i f i e d f o r m s o f n o r m a l l y o b s e r v e d t R N A s . T r e a t m e n t o f E. c o l i w i t h c h l o r a m p h e n i c o l c a u s e s t h e a c c u m u l a -Phe Phe t i o n o f n o v e l i s o a c c e p t o r s tRNA^ a n d tRNA.^ . Mann a nd Huang (175) h a v e p r e s e n t e d k i n e t i c e v i d e n c e t h a t d u r i n g r e c o v e r y f r o m Phe • Phe c h l o r a m p h e n i c o l t r e a t m e n t ; tRNA^. i s c o n v e r t e d t o tRNA^^ Phe w h i c h i n t u r n i s c o n v e r t e d t o t h e n o r m a l tRNA I f a r p r o p h a g e c a r r y i n g a tRNA gene i s i n d u c e d a b n o r m a l tRNA p r o f i l e s a r e o b t a i n e d f o r t h o s e t RNAs c a r r i e d . The new tRNA p e a k s a r e u n d e r m o d i f i e d t RNAs w h i c h a c c u m u l a t e b e c a u s e t h e p h a g e - c a r r i e d tRNA gene i s p r e s e n t a f t e r i n d u c t i o n i n v e r y l a r g e q u a n t i t i e s a n d t h e r e f o r e a b n o r m a l l y l a r g e q u a n t i t i e s o f t h i s tRNA a r e t r a n s c r i b e d . Hence t h e m o d i f i c a t i o n enzymes a r e r a t e l i m i t i n g i n t h e f o r m a t i o n o f m a t u r e tRNA. T h i s t e c h n i q u e h a s b e e n u s e d t o o b t a i n n o v e l tRNA i s o a c c e p t o r s i n many i n -s t a n c e s (26,176,177). The p h y s i o l o g i c a l s i g n i f i c a n c e o f t h e e x t r a p e a k s i s d i s c u s s e d i n t h i s t h e s i s i n t h e s e c t i o n d e a l i n g w i t h m i n o r b a s e f u n c t i o n . New i s o a c c e p t o r s a r e a l s o o b s e r v e d i f E. c o l i a r e s t a r v e d o f a r e q u i r e d a m ino e.g. r e f . (173,178-188). T h i s i s p a r t i c -u l a r l y a p p a r e n t i n t h e c a s e o f m e t h i o n i n e l i m i t a t i o n . I t h a s b e e n e s t a b l i s h e d i n t h e s e i n s t a n c e s t h a t t h e new tRNA i s o -a c c e p t o r s a r e d e f i c i e n t i n m o d i f i e d b a s e s . S t a r v a t i o n o f a m e v a l o n i c a c i d a u x o t r o p h f o r m e v a l o n i c a c i d g i v e s r i s e t o u n d e r -m o d i f i e d t R N A s (189). T h i s i s o b s e r v e d b e c a u s e m e v a l o n i c a c i d c a n a c t a s a p r e c u r s o r f o r p o r t i o n s o f c e r t a i n m o d i f i e d n u c l e o -s i d e s . F r o m t h e l i t e r a t u r e i t a p p e a r s t h a t many a d v e r s e g r o w t h c o n d i t i o n s are capable of g i v i n g r i s e to no v e l tRNA i s o a c c e p t o r s and t h a t s i m i l a r novel i s o a c c e p t o r s can be obt a i n e d from s e v e r a l growth c o n d i t i o n s . In o t h e r work w i t h E. c o l i , s t a r v a t i o n of a l e u c i n e o r tryptophan auxotroph was shown to have l i t t l e o r no e f f e c t on the t o t a l acceptance f o r the amino a c i d (190) absent from the medium. Repression o f the a r g i n y l b i o s y n t h e t i c pathway d i d not s i g n i f i c a n t l y a l t e r the arginyl-tRNA p r o f i l e ( 1 9 1 ) . Minor N u c l e o s i d e s i n tRNA In a d d i t i o n to the f o u r common RNA n u c l e o s i d e s tRNA has a wide v a r i e t y o f minor n u c l e o s i d e s c h a r a c t e r i z e d by the presence of unusual bases (hyper-modified forms of the common bas e s ) . In many i n s t a n c e s these minor bases have been shown t o p l a y an important r o l e i n tRNA f u n c t i o n s . The minor bases are c l a s s i f i e d i n t o t h r e e d i s t i n c t groups depending upon t h e i r l o c a t i o n i n the tRNA molecule. One group c o n s i s t s of those found i n the f i r s t p o s i t i o n o f the anticodon. A second group i n c l u d e s those l o c a t e d next to the 3'-OH end of the anticod o n . The t h i r d group c o n s i s t s of a l l minor bases not i n c l u d e d i n the f i r s t two. N e a r l y f o r t y m o d i f i e d n u c l e o s i d e s are known. For the purposes o f t h i s t h e s i s o n l y those found i n lower organisms and p a r t i c u l a r l y E. c o l i w i l l be d i s c u s s e d a t l e n g t h . The minor bases of the f i r s t grouping have r e c e i v e d con-s i d e r a b l e a t t e n t i o n because o f t h e i r a b i l i t y t o a l t e r codon-anticodon i n t e r a c t i o n s . The minor n u c l e o s i d e u r i d i n e - 5 - o x y a c e t i c a c i d occupies the f i r s t p o s i t i o n o f the anticodon o f E. c o l i tRNA^ a l ( 1 9 2 - 1 9 4 ) , E. c o l i tRNA^ e r (195) and i t s presence has b e e n s u g g e s t e d a t t h a t p o s i t i o n i n E. c o l i tRNA^"1"" (196) . U r i d i n e - 5 - o x y a c e t i c a c i d h a s u n i q u e c h a r a c t e r i s t i c s i n t h a t i t p a i r s w i t h U i n a d d i t i o n t o A a n d G ( 1 9 5 , 1 9 7 , 1 9 8 ) . I n t r i p l e t -V a l r i b o s o m e b i n d i n g s t u d i e s E. c o l i tRNA^ r e c o g n i z e d GUU w i t h 20% o f t h e e f f i c i e n c y w i t h w h i c h i t r e c o g n i z e d GUA a n d GUG (197) S e r S i m i l a r l y E. c o l i tRNA^ r e c o g n i z e d UCU w i t h 2 0 - 3 5 % o f t h e e f f i c i e n c y w i t h w h i c h i t r e c o g n i z e d UCA a n d UCG ( 1 9 5 , 1 9 8 ) . I n S e r a n i n v i t r o p r o t e i n s y n t h e s i z i n g s y s t e m E. c o l i tRNA^ was shown t o r e c o g n i z e t h e UCU c o d o n o f t h e f 2 c o a t p r o t e i n ( 1 9 9 ) . S t u d i e s o n E. c o l i t R N A A r g h a v e s u g g e s t e d t h a t i n o s i n e i s l o c a t e d i n t h e f i r s t p o s i t i o n o f t h e a n t i c o d o n ( 2 0 0 , 2 0 1 ) . I n o t h e r s y s t e m s i n o s i n e h a s b e e n shown t o b a s e p a i r w i t h U, C a n d A o f t h e t h i r d p o s i t i o n o f t h e c o d o n ( 2 0 2 , 2 0 3 ) . One s p e c i e s o f E. c o l i t R N A A r g r e c o g n i z e s t h e t r i p l e t s CGU, CGC and CGA. U r i d i n e - 5 - o x y a c e t i c a c i d a n d i n o s i n e h a v e i n common t h e a b i l i t y t o p a i r w i t h a b a s e w i t h w h i c h t h e u n m o d i f i e d f o r m s (U a n d A r e s p e c t i v e l y ) c a n n o t p a i r . t RNAs d e f i c i e n t i n t h e s e o d d b a s e s h a v e a more r e s t r i c t e d t r a n s l a t i o n a l c a p a c i t y . The m i n o r n u c l e o s i d e Q i s f o u n d i n t h e f i r s t p o s i t i o n o f t h e a n t i c o d o n i n E. c o l i t R NAs t h a t a c c e p t t y r o s i n e (3 09) h i s -t i d i n e , a s p a r a g i n e a n d a s p a r t i c a c i d ( 2 0 4 , 2 0 5 ) . A l l E. c o l i t R N As w h i c h r e c o g n i z e U a n d C i n t h e t h i r d p o s i t i o n and A i n t h e s e c o n d p o s i t i o n o f t h e c o d o n c o n t a i n Q ( 2 0 5 ) . Q i s a h i g h l y m o d i f i e d G a n d i s u n i q u e i n t h a t i t h a s a much g r e a t e r a f f i n i t y f o r U t h a n C. A number o f m i n o r n u c l e o s i d e s o c c u p y i n g t h e f i r s t p o s i t i o n o f t h e a n t i c o d o n h a v e b e e n shown t o be 2 - t h i o u r i d i n e d e r i v a t i v e s 28. 5 - M e t h y l ~ a m i n o m e t h y l - 2 - t h i o u r i d i n e h a s b e e n f o u n d t o o c c u p y t h a t p o s i t i o n i n E. c o l i t R N A ^ 1 1 1 (206,207) and t R N A G l n ( 2 0 8 ) . G l u S i m i l a r d e r i v a t i v e s h a v e b e e n f o u n d i n y e a s t tRNA^ (209) and r a t l i v e r t R N A ^ 1 1 1 a n d t R N A ^ 8 (210) . 2 - T h i o u r i d i n e d e r i v a t i v e s a r e u n i q u e i n t h a t t h e y show s t r i c t b a s e p a i r i n g w i t h A a n d n e v e r p a i r w i t h G. T h i s c h a r a c t e r i s t i c p r e v e n t s m i s c o d i n g i n s e v e r a l i n s t a n c e s (206).. T h o s e m i n o r n u c l e o s i d e s l o c a t e d i m m e d i a t e l y a d j a c e n t t o t h e 3'-OH e n d o f t h e a n t i c o d o n a r e l i k e w i s e v e r y i n t e r e s t i n g . Two s u c h n u c l e o s i d e s a r e 2 - m e t h y l t h i o - N 6 - ( A 2 - i s o p e n t e n y l ) adenosine(ms 2£ 6A) and N 6 - ( A 2 - i s o p e n t e n y l ) a d e n o s i n e ( i 5 A ) . I t h a s b e e n shown t h a t E. c o l i t R NAs w h i c h r e s p o n d t o a c o d o n s t a r t i n g w i t h U c o n t a i n one o f t h e s e b a s e s a t t h a t p o s i t i o n ( 2 1 1 - 2 1 3 ) . T h e s e n u c l e o s i d e s h a v e b e e n i d e n t i f i e d i n a number o f tRNAs o f o t h e r s y s t e m s . They h a v e b e e n i m p l i c a t e d i n r i b o -s o m a l b i n d i n g o f tRNA (176,214) i n t h a t when tRNAs w h i c h n o r m a l l y c o n t a i n t h e s e n u c l e o s i d e s a r e l a c k i n g them, t h e tRNA c a n no l o n g e r b i n d t o r i b o s o m e s e f f i c i e n t l y . t RNAs r e s p o n d i n g t o c o d o n s b e g i n n i n g w i t h U b u t l a c k i n g t h e s e m o d i f i e d n u c l e o s i d e s , c a n i n some s y s t e m s b i n d t o r i b o s o m e s w i t h n o r m a l e f f i c i e n c y ( 2 1 5 , 2 1 6 ) . N u c l e o s i d e s s u c h a s m s 2 £ 6 A a n d i 6 A h a v e b e e n shown t o h a v e s t r o n g c y t o k i n i n a c t i v i t y ( 2 5 , 2 1 7 ) . T h i s i s h o w e v e r n o t a p r o p e r t y d e p e n d e n t u p o n t h e i r p o s i t i o n o r p r e s e n c e i n tRNA ( 2 1 8 - 2 2 0 ) . C o n s i d e r a b l e w o r k h a s b e e n done t o d e t e r m i n e t h e m e c h a n i s m o f b i o s y n t h e s i s f o r t h e s e n u c l e o s i d e s (221-^223) . Phe A number o f tRNAs o f e u k a r y o t i c s y s t e m s h a v e a t t h i s p o s i t i o n t h e u n u s u a l n u c l e o t i d e Y o r a r e l a t e d d e r i v a t i v e 2 9 . ( 2 2 4 - 2 2 7 ) the s t r u c t u r e s o f which have been determined ( 2 2 7 -2 3 2 ) . These n u c l e o s i d e s appear necessary t o ma i n t a i n a p a r t i c u l a r conformation o f the anticodon loop ( 2 3 3 - 2 3 4 ) . The removal of Y Phe from t R N A y e a s t r e s u l t s i n the l o s s o f a b i l i t y of t h i s tRNA to p a r t i c i p a t e i n p r o t e i n s y n t h e s i s ( 2 3 5 ) . A group of h i g h l y m o d i f i e d n u c l e o s i d e s i n c l u d i n g N - [ 9 -( g - D - r i b o f u r a n o s y l ) p u r i n - 6 - y l - N - m e t h y l carbamoyl] t h r e o n i n e (mt 6A) and N - [ 9 - ( 3 - D - r i b o f u r a n o s y l ) p u r i n - 6 - y l - c a r b a m o y l ] t h r e o n i n e ( t 6 A ) was found t o occupy the p o s i t i o n immediately adjacent t o the 3'-OH end o f the an t i c o d o n i n tRNAs of E. c o l i which recog-n i z e codons s t a r t i n g w i t h A ( 2 3 6 - 2 3 8 ) . L i t t l e i s known about the f u n c t i o n o f these n u c l e o s i d e s although i t i s p o s t u l a t e d t h a t they p l a y a r o l e i n ribosome b i n d i n g s i m i l a r to t h a t d e s c r i b e d f o r the oth e r minor n u c l e o s i d e s a t t h a t p o s i t i o n . The mode of b i o s y n t h e s i s o f these n u c l e o s i d e s has been i n v e s t i g a t e d ( 2 3 9 - 2 4 1 ) . A number of o t h e r m o d i f i e d n u c l e o s i d e s have been i d e n t i f i e d a t t h i s p o s i t i o n . N 6-Methyl A i s u n i q u e l y found a d j a c e n t t o the 3'-0H end of the anticodon i n E. c o l i tRNA^ a l ( 1 9 2 / 1 9 3 ) . 1-Methyl G has been found a t t h a t p o s i t i o n o f E. c o l i t R N A L e u (CU s e r i e s ) ( 2 4 2 , 2 4 3 ) . 2-Methyl A has been i d e n t i f i e d a t t h a t p o s i t i o n i n the E. c o l i tRNA^ 1", t R N A A s p , tRNA^ l s and t R N A A r g ( 2 0 5 , 2 0 6 , 2 4 4 ) . The frequency o f such m o d i f i c a t i o n s a t t h i s p o s i t i o n i n tRNA suggests t h a t the m o d i f i e d bases p l a y a d e f i n i t e , perhaps c r i t i c a l r o l e , i n tRNA f u n c t i o n i n g . The t h i r d group of minor n u c l e o s i d e s i s v e r y d i v e r s e . Only s e l e c t e d examples w i l l be d i s c u s s e d here. The reader i s r e f e r r e d to s e v e r a l good sources o f a d d i t i o n a l i n f o r m a t i o n ( 1 6 , 2 1 , 2 5 ) . 30. Much of the early work done on the t h i r d group of minor nucleo-sides consisted of a cataloging of the kinds of modified nucleo-sides present and t h e i r location i n tRNA. More recent work has been concerned with the assignment of p a r t i c u l a r functions or properties to these nucleosides and with the understanding of how such minor nucleosides are involved i n o v e r a l l tRNA function. A good example of such work i s that described by Roe et a l . (245). They have c l e a r l y demonstrated that the N -methyl G i n tRNA at p o s i t i o n 10 from the 5 1 end dramatically affects the rate of aminoacylation by the cognate synthetase, increasing V max ten f o l d . As they point out, where minor nucleosides are shown to have such dramatic e f f e c t s on an important c e l l u l a r process, the presence or absence of such minor nucleosides constitutes a pot e n t i a l control mechanism over c e l l function and development. In other instances undermodified tRNA has been shown to have d i f f e r e n t charging c h a r a c t e r i s t i c s (246,247). This aspect of minor nucleoside function w i l l be considered again at length i n the Discussion portion of t h i s t h e s i s . Another possible function f o r methylated nucleosides has been suggested. Igo-Kemenes and Zachau (248) postulated that i t i s the function of the p o s i t i v e l y charged minor nucleosides such as 1-methyl A and 7-methyl G to s t a b i l i z e c e r t a i n regions of the tRNA structure by i n t e r a c t i o n with the negatively charged phosphates of adjacent regions. Another minor nucleoside of t h i s group which has been the subject of i n t e r e s t i n g research i s pseudouridine. In Salmonella, His tRNA of h i s t i d i n e T mutants was shown to have an altered c h r o m a t o g r a p h i c p r o f i l e . When t h e n u c l e o s i d e c o m p o s i t i o n o f H i s t R NA was e x a m i n e d i t was f o u n d t h a t t h e tRNA was l a c k i n g two p s e u d o u r i d i n e s i n t h e a n t i c o d o n arm. F u n c t i o n a l l y , t h e u n d e r -m o d i f i e d tRNA was u n a b l e t o p a r t i c i p a t e , a s t h e f u l l y m o d i f i e d f o r m d o e s , i n t h e r e p r e s s i o n o f t h e h i s t i d i n e b i o s y n t h e t i c p a t h -way (.310) . O t h e r t R N A s n o r m a l l y h a v i n g p s e u d o u r i d i n e i n t h e , a n t i c o d o n arm a l s o h a d a l t e r e d c h r o m a t o g r a p h i c p r o f i l e s (311) a n d w e r e u n a b l e t o p a r t i c i p a t e i n enzyme r e p r e s s i o n ( 2 5 0 ) . T h o s e p s e u d o u r i d i n e s f o u n d i n t h e common s e q u e n c e T^CG w e r e p r e s e n t s u g g e s t i n g t h a t s e v e r a l p s e u d o u r i d i n e f o r m i n g enzymes f o r tRNA e x i s t i n t h e c e l l . C o n v e r s i o n o f t h e u r i d i n e t o p s e u d o u r i d i n e i n tRNA h a s b e e n a c h i e v e d i n v i t r o ( 2 4 9 ) . A k e y r o l e i n t h e c o n t r o l o f c e l l u l a r m e t a b o l i s m c a n t h e r e f o r e be t e n t a t i v e l y a s s i g n e d t o t h e p s e u d o u r i d i n e s o f t h e a n t i c o d o n arm. The r e s u l t s o f m o s t o f t h e tRNA s t u d i e s t o d a t e s u g g e s t t h a t tRNA p o p u l a t i o n s a r e n o t j u s t random m i x t u r e s o f a v a r i e t y o f t R NAs b u t a r e s p e c i f i c p o p u l a t i o n s o f p a r t i c u l a r i s o a c c e p t o r s . T h i s i m p l i e s t h a t s e n s i t i v e c o n t r o l m e c h a n i s m s a r e o p e r a t i v e w h i c h c a n m e a s u r e t h e amounts o f i n d i v i d u a l t RNAs p r e s e n t a n d w h i c h c a n b r i n g a b o u t s p e c i f i c q u a n t i t a t i v e a n d q u a l i t a t i v e c h a n g e s i n t h e c e l l u l a r tRNA p o p u l a t i o n . I n E. c o l i t h e r e l g e n e c o n t r o l o f t o t a l t RNA b i o s y n t h e s i s h a s b e e n s t u d i e d e x t e n s i v e l y . We h y p o t h e s i z e t h a t a d d i t i o n a l more s e l e c t i v e c o n t r o l m e c h a n i s m s a r e o p e r a t i v e t o r e g u l a t e i n d i v i d u a l tRNA b i o s y n t h e s i s . I n t h i s t h e s i s , an a t t e m p t h a s b e e n made t o d e m o n s t r a t e s p e c i f i c tRNA b i o s y n t h e s i s i n E. c o l i i n r e s p o n s e t o a d v e r s e g r o w t h c o n d i t i o n s . A s a s t a r t i n g p o i n t we h a v e s o u g h t t o t e s t t h e h y p o t h e s i s t h a t i n d i v i d u a l t RNAs a r e i n v o l v e d i n r e g u l a t i o n o f tRNA b i o s y n t h e s i s a c t i n g a s f e e d -b a c k r e p r e s s o r s a t t h e t r a n s c r i p t i o n a l l e v e l . T h i s h y p o t h e s i s h a s n o t b e e n a d e q u a t e l y t e s t e d i n t h e l i t e r a t u r e . T h e r e i s c o n -s i d e r a b l e e v i d e n c e (315-319) t h a t c o n f o r m a t i o n a l c h a n g e s i n tRNA accompany a m i n o a c y l a t i o n . We h y p o t h e s i z e t h a t a m i n o a c y l a t i o n t h u s c o n v e r t s a n i n a c t i v e tRNA r e p r e s s o r t o t h e a c t i v e f o r m . I n d i v i d u a l a m i n o a c y l a t e d tRNAs c o u l d t h e n s p e c i f i c a l l y f e e d b a c k i n h i b i t t r a n s c r i p t i o n o f t h o s e p a r t i c u l a r tRNA g e n e s . T h i s h y p o t h e s i s d o e s n o t r e q u i r e t h a t a l l tRNA b i o s y n t h e s i s w o u l d be c o n t r o l l e d i n t h i s manner. S e v e r a l p o s s i b i l i t i e s e x i s t and w i l l be d i s c u s s e d . The f i r s t e x p e r i m e n t s i n v o l v e d t h e a l t e r a t i o n o f s p e c i f i c a m i n o a c y l - t R N A l e v e l s b y a m ino a c i d a n a l o g u e s o r a m i no a c i d d e p l e t i o n a n d t h e a n a l y s i s o f t h e tRNA p o p u l a t i o n s o f t h e E. c o l i u n d e r t h e s e g r o w t h c o n d i t i o n s . The h y p o t h e s i s p r e s e n t e d p r e d i c t s t h a t an i n c r e a s e d t r a n s c r i p t i o n o f tRNA w i l l b e o b s e r v e d f o r t h o s e tRNAs w h i c h a r e f e e d b a c k i n h i b i t e d b y a p a r t i c u l a r a m i n o a c y l - t R N A . M a t e r i a l s The f o l l o w i n g a m ino a c i d a n a l o g u e s w e r e p u r c h a s e d f r o m C a l b i o c h e m : D L - 2 - a m i n o - n - b u t y r a t e , D L - p - f l u o r o - p h e n y l a l a n i n e , a n d l - a m i n o - 2 - ( 4 - h y d r o x y p h e n y l ) e t h y l p h o s p h o n a t e . The a n a l o g u e O - m e t h y l - D L - t h r e o n i n e was p u r c h a s e d f r o m C y c l o C h e m i c a l Co. The m a t e r i a l s f o r RPC-5 c o l u m n s , P l a s k o n CTFE 2300 powder and A d o g e n 4 6 4 , w e r e o b t a i n e d f r o m A l l i e d C h e m i c a l C o r p . and A s h l a n d C h e m i c a l Co. r e s p e c t i v e l y . RPC-5 was p r e p a r e d a s d e s c r i b e d i n t h e l i t e r a t u r e ( 1 2 4 , 2 5 1 ) . S e p h a d e x G-25 was o b t a i n e d f r o m P h a r m a c i a . P a n c r e a t i c RNase, RNase T 2 , R N a s e T^, s n a k e venom p h o s p h o d i e s t e r a s e a n d E. c o l i a l k a l i n e p h o s p h a t a s e w e r e p u r c h a s e d f r o m W o r t h i n g t o n B i o c h e m i c a l s . C e l l u l o s e t h i n l a y e r c h r o m a t o g r a p h i c s h e e t s w e r e p u r c h a s e d f r o m E a s t m a n Kodak Co. T h e s e w e r e w i t h o u t f l u o r e s c e n t i n d i c a t o r (no . 1 3 2 5 5 ) . V a l P u r i f i e d tRNA^ , (1500 p i c o m o l e s o f v a l i n e a c c e p t a n c e p e r A 2 6 o u n i t ) was a k i n d g i f t f r o m D r . M i l d r e d Cohn. C r u d e c o m m e r c i a l tRNA was o b t a i n e d f r o m S c h w a r z - M a n n Co. A l l common c h e m i c a l s , u n l a b e l l e d amino a c i d s e t c . w e r e o b -t a i n e d c o m m e r c i a l l y a n d w e r e o f r e a g e n t g r a d e . A l l r a d i o a c t i v e a m ino a c i d s w e r e p u r c h a s e d f r o m New E n g l a n d N u c l e a r C o r p . P o t a s s i u m b o r o h y d r i d e - [ 3 H ] 7.6 C i / m m o l e was p u r -c h a s e d f r o m A m e r s h a m / S e a r l e . The c o u n t i n g c o c k t a i l , A q u a s o l , was p u r c h a s e d f r o m New E n g l a n d N u c l e a r C o r p . E. c o l i NF162 ( A r g ~ M e t " r e l ~ ) a n d E. c o l i NF161 ( A r g ~ M e t " r e l + ) w e r e g i f t s f r o m D r . J . G a l l a n t . E. c o l i B was o b t a i n e d f r o m D r . R . A . J . W a r r e n . E. c o l i B stru, a n a d d i t i o n a l E. c o l i B s t r a i n a n d E. c o l i W w e r e o b t a i n e d f r o m D r . W.J. P o l g l a s e . F r o z e n c o m m e r c i a l E. c o l i ( m i d l o g ) was p u r c h a s e d f r o m G r a i n P r o c e s s i n g Co. M e t h o d s G r o w t h o f E. c o l i The v a r i o u s E. c o l i s t r a i n s w e r e g r o w n o n 0.4% g l u c o s e p l u s m i n i m a l s a l t s medium ( 2 5 2 ) . The medium f o r NF161 a n d NF162 was s u p p l e m e n t e d w i t h 50 ug p e r m l o f e a c h o f t h e r e q u i r e d a m ino a c i d s E. c o l i B s t r D was g r o w n i n t h e p r e s e n c e o f 1 mg p e r m l o f d i -h y d r o s t r e p t o m y c i n s u l f a t e . T u r b i d i t y was m o n i t o r e d a t 420 nm on t h e a b s o r b a n c e s c a l e u s i n g a B a u s c h a n d Lomb S p e c t r o n i c 2 0 , f r o m a n i n i t i a l t u r b i d i t y o f 0.05 t o a f i n a l v a l u e o f 0.95. L o g a r i t h m i c a l l y g r o w i n g c e l l s w e r e u s e d a s i n o c u l u m t o m i n i m i z e g r o w t h l a g i n t h e e x p e r i m e n t a l c u l t u r e . G r o w t h r a t e was e x -p r e s s e d i n t e r m s o f t h e number o f d o u b l i n g s o f t u r b i d i t y p e r h o u r . I n l a r g e s c a l e p r e p a r a t i o n s t h e b a c t e r i a w e r e g r o w n a t 37°, u n l e s s o t h e r w i s e n o t e d , i n 20 1. b a t c h e s i n an A m e r i c a n S t e r i l i z e r B i o g e n u n d e r a e r o b i c c o n d i t i o n s (15 p . s . i . a i r p r e s s u r e , 142 rpm a g i t a t o r s p e e d a nd s e t t i n g 2 o n t h e B i o g e n f l o w m e t e r ) . B e c a u s e o f t e c h n i c a l p r o b l e m s t h e g r o w t h medium was n o t s t e r i -l i z e d b u t c o n t a m i n a t i o n was m o n i t o r e d a nd i f more t h a n 4% o f t h e c e l l s w e r e c o n t a m i n a n t s t h e r u n was d i s c a r d e d ( s e e D i s c u s s i o n ) . C e l l s w e r e h a r v e s t e d u s i n g a S h a r p i e s c o n t i n u o u s f l o w c e n t r i f u g e . I n s m a l l p r e p a r a t i o n s , c e l l s w e r e g r o w n a s p r e v i o u s l y d e s -c r i b e d i n 250 m l v o l u m e s i n 11'. e r l e n m e y e r f l a s k s w i t h s h a k i n g i n a New B r u n s w i c k S c i e n t i f i c Co. w a t e r b a t h s h a k e r . T h e s e c e l l s w e r e t h e n h a r v e s t e d i n a S o r v a l l RC-2B c e n t r i f u g e . The i d e n t i t i e s o f NF161 and NF162 c e l l s w e r e e a s i l y c o n -f i r m e d b y t h e y i e l d o f tRNA a n d by t h e i r i n a b i l i t y t o g r o w o n u n s u p p l e m e n t e d m e d i a . I n e x p e r i m e n t s i n w h i c h E. c o l i B w e r e g r o w n a t e x t r e m e t e m p e r a t u r e s t h e c e l l s a t t h e t i m e o f h a r v e s t -i n g w e r e p l a t e d o u t t o c h e c k f o r c o n t a m i n a t i o n . S e l e c t e d p l a t e d c o l o n i e s w e r e a l s o c h e c k e d f o r s e n s i t i v i t y t o p h a g e T7. The s t r a i n s w e r e m a i n t a i n e d i n a p p r o p r i a t e m e d i a i n 50 m l v o l u m e s i n 125 m l e r l e n m e y e r f l a s k s i n a w a t e r b a t h s h a k e r a t 37° o r o n 1.5% a g a r p l a t e s c o n t a i n i n g t h e medium r e q u i r e d f o r g r o w t h o f t h a t s t r a i n . I n e x p e r i m e n t s w h e r e E. c o l i NF162 was s t a r v e d f o r a r g i n i n e , 13.4 ug/ml o f a r g i n i n e a n d 50 ug/ml o f m e t h i o n i n e w e r e a d d e d . I n t h o s e e x p e r i m e n t s w h e r e E. c o l i NF162 was s t a r v e d f o r m e t h i o -n i n e 2.1 y g / m l o f m e t h i o n i n e a n d 50 ug/ml o f a r g i n i n e w e r e a d d e d . I n e a c h i n s t a n c e t h e c h o s e n a mino a c i d became g r o w t h l i m i t i n g s u c h t h a t a f t e r t h r e e h o u r s o f s t a r v a t i o n t h e t u r b i d i t y a t 420 nm was 0.95. A l l g r o w t h i n h i b i t o r s w e r e a d d e d a t t h e t i m e o f i n o c u l a t i o n e x c e p t a - a m i n o - n - b u t y r a t e w h i c h was a d d e d when t h e t u r b i d i t y a t 420 nm r e a c h e d 0.50. P r e p a r a t i o n o f A m i n o a c y l - t R N A S y n t h e t a s e s C r u d e E. c o l i a m i n o a c y l - t R N A s y n t h e t a s e s w e r e p r e p a r e d f r o m c o m m e r c i a l l y g r o w n c e l l s ( m i d l o g ) e s s e n t i a l l y b y t h e m e t h o d o f Muench a n d B e r g (253) e x c e p t t h a t t h e c e l l s w e r e d i s r u p t e d u s i n g a P o l y t r o n ( K i n e m a t i c a GMBH, S w i t z e r l a n d ) a n d a f t e r t h e DEAE-c e l l u l o s e c h r o m a t o g r a p h y s t e p , t h e s y n t h e t a s e p r e p a r a t i o n was d i a l y z e d a g a i n s t T r i s - H C l (pH 7.4) 20 mM; 2 - m e r c a p t o e t h a n o l 10 mM; 50% g l y c e r o l . The p r o d u c t was s t o r e d a t -20°C. The g l y c e r o l was r e m o v e d b e f o r e t h e a m i n o a c y l a t i o n r e a c t i o n s b y c h r o m a t o g r a p h y on S e p h a d e x G-25. P r e p a r a t i o n o f tRNA C r u d e E. c o l i tRNA was p r e p a r e d by t h e K i r b y p h e n o l m ethod (254) f o l l o w e d b y D E A E - c e l l u l o s e c h r o m a t o g r a p h y . E. c o l i was h o m o g e n i z e d i n 2 v o l u m e s o f 88% p h e n o l and b u f f e r A. B u f f e r A c o n t a i n e d 10 mM T r i s - H C l (pH 7 . 5 ) , 10 mM m a g n e s i u m c h l o r i d e a n d 1 mM 2 - m e r c a p t o e t h a n o l . The h o mogenate was c e n t r i f u g e d a t 10,000 X g f o r 15 m i n a n d t h e a q u e o u s l a y e r r e m o v e d . The p h e n o l l a y e r was r e - e x t r a c t e d w i t h 1 v o l u m e o f b u f f e r A a n d t h e h o mogenate was c e n t r i f u g e d a s b e f o r e . The c o m b i n e d a q u e o u s f r a c t i o n s w e r e m i x e d w i t h 0.1 v o l u m e o f 2.0 M p o t a s s i u m a c e t a t e (pH 4 . 5 ) , t h e n w i t h 2.5 v o l u m e s o f 95% e t h a n o l and l e f t o v e r n i g h t a t -20°C. The n u c l e i c a c i d p r e c i p i t a t e 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 , r e s u s p e n d e d i n b u f f e r A a n d a p p l i e d t o a D E A E - c e l l u l o s e c o l u m n p r e v i o u s l y e q u i l i b r a t e d w i t h b u f f e r A. F o r s m a l l p r e p a r a t i o n s t h e n u c l e i c a c i d p r e c i p i t a t e s w e r e c o l l e c t e d o n M i l l i p o r e f i l t e r s a n d t h e n h a n d l e d s u b s e q u e n t l y a s d e s c r i b e d b e l o w . The c o l u m n was w a s h e d w i t h b u f f e r A c o n t a i n i n g 0.3 M N a C l and t h e c r u d e tRNA e l u t e d w i t h 1.0 M N a C l i n b u f f e r A. The tRNA was p r e c i p i t a t e d w i t h e t h a n o l a n d c o l l e c t e d b y c e n t r i f u g a t i o n a f t e r b e i n g s t o r e d o v e r n i g h t a t -20°C. I t was t h e n d i s s o l v e d i n d i s t i l l e d w a t e r , d i a l y z e d a g a i n s t d i s t i l l e d w a t e r a n d f r e e z e - d r i e d . A m i n o a c y l a t i o n . o f tRNA. A c c e p t a n c e L e v e l s E x c e p t w h e r e n o t e d i n p a r t i c u l a r e x p e r i m e n t s , a m i n o a c y l a -38. tions were c a r r i e d out at 21°C i n reaction volumes of 0.20 ml. Each reaction contained per ml, T r i s HC1 (pH 7.4), 50 ymoles; 2-mercaptoethanol, 5 ymoles; 1^C-labelled amino acid, 50 nmoles (20 yCi/ymole); MgCl2, 10 ymoles; ATP, 10 ymoles, 3 to 5 A 2eo units of crude E. c o l i tRNA and s u f f i c i e n t crude aminoacyl-tRNA synthetase to achieve complete charging within 5 min. To follow the progress of the reaction, 50 y l aliquots were pipetted onto f i l t e r paper discs and the t r i c h l o r o a c e t i c acid-insoluble radio-active amino acids determined. Amino acid acceptance values were expressed r e l a t i v e to an in t e r n a l standard, the acceptance of threonine. Measurements were considered v a l i d i f amino acid acceptance was d i r e c t l y proportional to tRNA added and i f amino acid acceptance at 5, 10 and 20 minutes of incubation were i d e n t i c a l , thus in d i c a t i n g rapid saturation of the acceptor. Aminoacylation of tRNA for RPC-5 Chromatography Aminoacyl-tRNA for chromatography on RPC-5 was prepared e s s e n t i a l l y as described to obtain acceptance l e v e l s except that the l a b e l l e d amino acids were of higher s p e c i f i c a c t i v i t y and were present at concentrations of 10-3 0 nmoles per ml and 0.4 ml reaction volumes were used. The amounts of synthetase and tRNA were scaled up proportionately. The la b e l l e d aminoacyl-tRNAs thus prepared were then chromatographed on DEAE-cellulose as described by Yang and N o v e l l i (255) to obtain an aminoacyl-tRNA f r a c t i o n free of protein and undesirable small molecules. Aminoacyl-tRNAs were stored i n the eluti o n buffer at -20°C. RPC-5 C h r o m a t o g r a p h y R a d i o a c t i v e l y l a b e l l e d a m i n o a c y l - t R N A s w e r e c h r o m a t o g r a p h e d o n t h e RPC-5 s y s t e m d e v e l o p e d by P e a r s o n e t a J . ( 2 5 1 ) . RPC-5 c o l u m n s w e r e p r e p a r e d and r u n a s d e s c r i b e d b y W h i t e e t a l . ( 1 2 4 ) . E l u t i o n was w i t h l i n e a r N a C l g r a d i e n t s i n a b u f f e r c o n t a i n i n g 5 mM s o d i u m a c e t a t e (pH 4 . 5 ) , 10 mM MgCl2 a n d 1 mM 2 r m e r c a p t o e t h a n o l . A n a l y t i c a l c o l u m n s w e r e 12 t o 15 cm l o n g a nd j a c k e t e d (37°C). As many a s 18 A 2 6 0 u n i t s o f a m i n o a c y l - t R N A w e r e c h r o m a t o g r a p h e d e a c h t i m e . C o l u m n s w e r e n o r m a l l y r e p l a c e d a f t e r 2 0 s u c h r u n s o r a s s o o n a s t h e y showed s i g n s o f d e t e r i o r a t i o n ( a b n o r m a l p e a k w i d t h ) . T h e s e c o l u m n s w e r e e l u t e d a t 15 m l / h r w i t h a 100 m l g r a d i e n t . The 0.5 m l f r a c t i o n s w e r e s h a k e n w i t h 7 v o l u m e s o f A q u a s o l a n d r a d i o a c t i v i t y was d e t e r m i n e d i n a s c i n t i l l a t i o n c o u n t e r . To o b t a i n l a b e l l e d a m i n o a c y l - t R N A i s o a c c e p t o r s f o r 3'OH e n d a n a l y s i s , a s many a s 40 A 2 6 o u n i t s o f l a b e l l e d a m i n o a c y l -tRNA w e r e c h r o m a t o g r a p h e d o n a 60 cm X 0.9 cm c o l u m n . T h e s e c o l u m n s w e r e e l u t e d a t 40 m l / h r w i t h a 200 m l g r a d i e n t . R a d i o -a c t i v i t y was d e t e r m i n e d i n 50 t o 100 u l o f t h e 1 m l f r a c t i o n s a n d t h e d e s i r e d f r a c t i o n s w e r e p o o l e d . V a l RPC-5 was a l s o u s e d i n t h e p u r i f i c a t i o n o f t R N A 3 . The d e t a i l s a r e i n c l u d e d b e l o w . V a l P u r i f i c a t i o n o f t R N A 3 S e v e r a l b a t c h e s o f E. c o l i NF162 w e r e g r o w n u n d e r c o n d i t i o n s o f l i m i t i n g a r g i n i n e a s p r e v i o u s l y d e s c r i b e d . The tRNA was i s o -l a t e d a nd p o o l e d . The r e s u l t i n g 1.90 g o f c r u d e tRNA was u s e d 40. for the i s o l a t i o n of p u r i f i e d tRNA*01"'". The p u r i f i c a t i o n of Val tRNA^ involved standardized methods and thus w i l l only be d i s -cussed b r i e f l y . The f i n a l p u r i f i c a t i o n step i s described i n d e t a i l so that the reader w i l l be aware of the purity of the material used i n subsequent experiments. For convenience, the tRNA was divided into approximately equal portions. Each portion was aminoacylated and naphthoxy-acetylated by standard procedures (263,264). The derivatized material was chromatographed on BD-cellulose and the f r a c t i o n eluted with 1.0 M NaCl plus 20% ethanol, but not by 1.0 M NaCl plus 5% ethanol, was i s o l a t e d . The s a l t fractions were reamino-acylated, derivatized, run on BD-cellulose and the 1.0 M NaCl plus 20% ethanol f r a c t i o n was pooled with that from the f i r s t Val i s o l a t i o n . The naphthoxylacetylated Val-tRNA was hydrolysed i n Va 1 1.8 M T r i s buffer (pH 8.0) and the tRNA isola t e d i n the s a l t f r a c t i o n on BD-cellulose. The tRNA V a l was then run on a 2.4 x 4.5 cm RPC-5 column (50 mesh sieve size) and eluted with standard RPC-5 buffers (pH 4.5) within a l i n e a r NaCl gradient from 0.50 to 0.65 M. [ 1^C]Valine acceptance was determined by charging using f i l t e r paper discs on a p l a s t i c support (266). The appropriate fractions were pooled and rerun on the same column under the same conditions. Again the [ l l fC]valine accep-tance was determined and the appropriate fractions pooled. This material (47.8 A 2 6 o units) was then applied to the RPC-5 column described and eluted with a l i n e a r gradient (2 1.) of NaCl from 0.50 to 0.65 M i n 10 mM Tris-HCl pH 7.0, 1 mM 2-mercaptoethanol. The f r a c t i o n s i z e was 10 m l and t h e c o l u m n f l o w r a t e 18 0 m l / h r . T h i s c o l u m n was t h e f i n a l s t e p i n t h e p u r i f i c a t i o n a nd t h e r e s u l t s a r e d e s c r i b e d i n F i g . 40. P o l y a c r y l a m i d e G e l E l e c t r o p h o r e s i s To a n a l y z e RNA s i z e d i s t r i b u t i o n b y p o l y a c r y l a m i d e g e l e l e c t r o p h o r e s i s t h e f o l l o w i n g p r o c e d u r e s w e r e u s e d . S t o c k TEB b u f f e r (256) c o n t a i n e d 108 g T r i s b a s e , 9.3 g EDTA, d i s o d i u m s a l t a n d 55 g b o r i c a c i d p e r l i t r e . A n a l y t i c a l g e l s l a b s 3 mm t h i c k w e r e c a s t i n a c e l l (257) p u r c h a s e d f r o m t h e E-C A p p a r a t u s Co., S t . P e t e r s b u r g , F l o r i d a . The g e l s w e r e made f r o m 1 4 . 7 % a c r y l a m i d e and 0.3% m e t h y l e n e b i s a c r y l a m i d e i n 6 M u r e a c o n a i n i n g 2% o f s t o c k TEB b u f f e r . The s t a c k i n g g e l , i n w h i c h t h e s a m p l e s l o t s w e r e c a s t , was 4.8% a c r y l a m i d e , 0.2% m e t h y l e n e b i s a c r y l a m i d e i n 2% s t o c k TEB b u f f e r ( w i t h o u t u r e a ) . The l o w e r r e s e r v o i r was f i l l e d w i t h 2% s t o c k TEB b u f f e r i n 6 M u r e a a n d t h e u p p e r w i t h 2.0% s t o c k TEB b u f f e r w i t h o u t u r e a . The g e l was p r e r u n f o r 30-60 m i n u t e s and e l e c t r o d e c o m p a r t m e n t s r e f i l l e d w i t h f r e s h b u f f e r . S a m p l e s o f RNA c o n t a i n i n g a b o u t 0.5 A 2 6 0 u n i t s i n 10 t o 20 u l o f w a t e r w e r e m i x e d w i t h 0.5 t o 1 v o l u m e o f 50% s u c r o s e s o l u t i o n ( r i b o n u c l e a s e f r e e ) a n d 1 u l o f 0.001% b r o m o p h e n o l b l u e s o l u t i o n a n d t r a n s f e r r e d t o t h e 1 cm w i d e s a m p l e s l o t s . E l e c t r o p h o r e s i s was p e r f o r m e d i n t h e c o l d room (4°) w i t h w a t e r (6°) c i r c u l a t i n g t h r o u g h t h e c o o l i n g j a c k e t . A t 15 V/cm i t r e q u i r e d 6 h r f o r t h e t r a c k i n g d y e t o m i g r a t e t o t h e b o t t o m o f t h e g e l . The d e v e l o p e d g e l was r e m o v e d , s t a i n e d i n S t a i n s - A l l (258) o v e r n i g h t a t 37° a n d d e s t a i n e d i n w a t e r by b l e a c h i n g w i t h l i g h t . RNA m a r k e r s w e r e o b t a i n e d f r o m c o m m e r c i a l tRNA o f E. c o l i B ( S c h w a r z / M a n n , O r a n g e b u r g , New Y o r k ) b y e l e c t r o -p h o r e s i s i n a 1 2 % g e l ( 1 1 . 6 5 % a c r y l a m i d e a n d 0.35% m e t h y l e n e -b i s a c r y l a m i d e ) i n 2% s t o c k TEB b u f f e r (no u r e a ) . Ten A 2 6 0 ' u n i t s o f c r u d e RNA w e r e l o a d e d i n t o t h e s i n g l e s l o t 10 cm w i d e . The d e v e l o p e d g e l was l i g h t l y s t a i n e d a n d t h e RNA s a m p l e s r e c o v e r e d a s d e s c r i b e d by C h i a e t a l . ( 2 5 9 ) . T h e s e p r o c e d u r e s w e r e c a r r i e d o u t , a s i n d i c a t e d i n t h e a c k n o w l e d g e m e n t s , by D r . I.C. G i l l a m . N u c l e o s i d e A n a l y s i s o f P u r i f i e d t RNAs T o t a l n u c l e o s i d e a n a l y s i s was p e r f o r m e d u s i n g t h e R a n d e r a t h t r i t i u m d e r i v a t i v e m e t h o d (260) w i t h t h e m o d i f i c a t i o n s t h a t KBH^ i n KOH was u s e d and t h e t h i n l a y e r c h r o m a t o g r a p h y was p e r -f o r m e d u s i n g s o l v e n t s y s t e m s F and G ( 2 6 0 ) . A q u a l i t a t i v e a n d q u a n t i t a t i v e c o m p a r i s o n o f t h e n u c l e o s i d e c o m p o s i t i o n o f p u r i f i e d t R N A ^ a l a n d tRNA^a"'' was made. The p u r i f i e d tRNA s a m p l e s w e r e d i g e s t e d i n a t o t a l r e a c t i o n v o l u m e o f 50 u l u s i n g 1.0 A 2 6 o u n i t o f p u r i f i e d RNA p e r d i g e s t i o n . The [ 3 H ] p o t a s s i u m b o r o h y d r i d e u s e d h a d a s p e c i f i c a c t i v i t y o f 7.6 C i /mmole when p u r c h a s e d . The c h r o m a t o g r a m s w e r e s p o t t e d w i t h a p p r o x i m a t e l y 0.06 A 2 6 o o f t h e l a b e l l e d n u c l e o s i d e s . The a r e a s o f t h e l a b e l l e d n u c l e o s i d e t r i o l s a s d e d u c e d f r o m t h e f l u o r o g r a m s w e r e c u t o u t a n d e l u t e d i n 4 m l o f 2 M ammonium h y d r o x i d e f o r a p p r o x i m a t e l y 8 h r s . T h i s l e n g t h o f t i m e was n e c -e s s a r y f o r c o m p l e t e e l u t i o n o f t h e t r i o l f r o m t h e c h r o m a t o g r a p h y p l a t e r a t h e r t h a n t h e 2 h o u r s e l u t i o n t i m e s u g g e s t e d i n t h e l i t e r -a t u r e . F rom t h e r a d i o a c t i v i t y o f t h e e l u a t e t h e n u c l e o s i d e com-p o s i t i o n was c a l c u l a t e d a s s u m i n g a tRNA l e n g t h o f 7 6 n u c l e o s i d e r e s i d u e s . 43. Qualitative Nucleotide Analysis of .tKNA^ *1"1" Val Three A260 units of p u r i f i e d tRNA3 was digested overnight at 37°C i n a t o t a l volume of 5.0yl containing 10 y l of RNase T 2 and 10 y l of 0.1 M potassium acetate pH 4.3. The digestion products were spotted on c e l l u l o s e thin layer chromatography plates and developed as described by Nishimura (16). Nucleotide positions were determined by UV absorption and by fluorescence afte r exposure to HC1 vapors. [ 3H]Val-Oligonucleotides on DEAE-Cellulose The procedure i s b a s i c a l l y that of Twardzic et a l . (265). Individual [ 3H]Val-tRNA^ 1 isoacceptors were prepared as pre-viously described, suspended i n 0.10 M potassium acetate (pH 4.5), 2 mM disodium EDTA and incubated with RNase T-^ . The t o t a l digest was applied to a DEAE-cellulose column (1.2 cm x 15 cm) which had been pre-equilibrated with 10 mM ammonium formate (pH 4.5). The column was eluted with a l i n e a r ammonium formate gradient, t o t a l volume 360 ml, from 10 mM to 0.25 M ammonium formate Va 1 (pH 4.5). Undigested Val-tRNA was then eluted with 1.0 M ammonium formate (pH 4.5). The column flow rate was 30 ml/hr. and the f r a c t i o n size was 2.0 ml. An aliquot of 0.4 ml from every second tube was counted. Results and Discussion The theory proposed i n the introduction as a s t a r t i n g point for the investigation of the control of tRNA biosynthesis i n E. c o l i would predict that the presence of amino acid analogues which l i m i t aminoacylation or the absence of a required amino acid, would bring about s p e c i f i c derepression of the synthesis of tRNA for which there was a decreased l e v e l of aminoacyl-tRNA. This should r e s u l t i n increased amino acid acceptance of the is o l a t e d tRNA for that tRNA having the altered aminoacylation l e v e l . Although aminoacyl-tRNA l e v e l s were not determined i n vivo, i t i s well established (267,289) that amino acid ana-logues and amino acid starvation do lead to correspondingly decreased aminoacyl-tRNA l e v e l s and i t i s assumed that such was the case i n the experiments described here and that t h i s resulted i n the reduced growth rates observed. Amino Acid Analogues I n i t i a l l y 26 amino acid analogues were examined to determine or confirm t h e i r a b i l i t y to i n h i b i t E. c o l i growth and amino-acylation i n v i t r o . Based upon the growth i n h i b i t i o n r e s u l t s obtained, r e s u l t s published i n the l i t e r a t u r e , the a v a i l a b i l i t y of the analogues etc. i t was decided that the analogues l i s t e d i n Table 1 were the most suitable analogues for the subsequent experiments. The isoleucine analogue, O-methyl-DL-threonine, was shown to be a potent growth i n h i b i t o r of E. c o l i under the growth con-d i t i o n s described. In subsequent growth experiments i n the 45. T a b l e 1 E f f e c t o f Amino A c i d A n a l o g u e s o n E. c o l i G r o w t h A m ino A c i d A n a l o g u e s G r o w t h R a t e d.p.h. Maximum Concen-t r a t i o n u s e d Comments C o n t r o l - B 0- m e t h y l - D L t h r e o n i n e 1- a m i n o - 2 - ( 4 -h y d r o x y p h e n y 1 ) e t h y l p h o s p h o n a t e D L - p - f l u o r o p h e n y l a l a n i n e D L - a - a m i n o - n b u t y r a t e 1.0 0.6 t o no g r o w t h 0.8 0.55 0.50 10 t o 300 y g / m l 100 y g / m l 15 y g / m l 200 y g / m l i s o l e u c i n e a n a l o g u e t y r o s i n e a n a l o g u e p h e n y l a l a n i n e a n a l o g u e v a l i n e a n a l o g u e E. c o l i w e r e grown i n 50 m l v o l u m e s i n 125 m l f l a s k s w i t h v i g o r -ous s h a k i n g i n a 37°C w a t e r b a t h , a s d e s c r i b e d i n M e t h o d s , i n t h e p r e s e n c e o f i n d i v i d u a l a m i n o a c i d a n a l o g u e s . G r o w t h was m o n i t o r e d b y t u r b i d i t y m e a s u r e m e n t s a t 420 nm. Biogen i t was far less growth i n h i b i t o r y . The a b i l i t y of an amino acid analogue to be an i n h i b i t o r of E. c o l i growth depends very much on the growth conditions used as can be seen by compar-ing growth rates i n the presence of the analogues i n Tables 1 and 2. O-Methyl-DL-threonine has been shown by other workers (312,313) to i n h i b i t E. c o l i growth, to i n h i b i t isoleucine aminoacylation, to be incorporated into protein and to compet-i t i v e l y i n h i b i t threonine deaminase feedback i n h i b i t i o n (313). A large number of phosphonic acids were shown to i n h i b i t E. c o l i growth at suitable concentrations. Only the tyrosine phosphonate, l-amino-2-(4-hydroxyphenyl)ethyl phosphonate was capable of i n h i b i t i n g aminoacylation i n v i t r o . By use of a Dixon plot the tyrosine phosphonate was shown to be a competitive i n h i b i t o r of [ 1^C]Tyr-tRNA T y r formation having a Ki of approx-imately 10 h M under the acylation conditions used and described i n Methods. Several amino acid phosphonate analogues have been shown to i n h i b i t aminoacylation i n other organisms (314,276). The phenylalanine analogue DL-p-fluorophenylalanine was shown to i n h i b i t E. c o l i growth at r e l a t i v e l y low concentrations. I t has been demonstrated by many other workers that p-fluoro-phenylalanine i s a potent E. c o l i growth i n h i b i t o r (277-283) that i t i s incorporated into protein (278-284) i n h i b i t s amino-Phe acylation by phenylalanine (285,28 6) of tRNA and i s an i n h i b i t o r of phenylalanine biosynthetic enzymes (287). The valine analogue, DL-a-amino-n-butyrate was shown to be growth i n h i b i t o r y atvery high analogue concentrations. This analogue has. been shown by others to i n h i b i t E. c o l i growth (288) and acylation of tRNA v a x by valine but not to be acylated i t s e l f (289) . Of the four analogues i n Table 1, two are incor-porated under cert a i n conditions into protein (p-fluorophenyl-alanine and O-methyl-threonine) and two are not (a-amino-n-butyrate and the tyrosine phosphonate). tRNA of E. c o l i Treated with Amino Acid Analogues and of E. c o l i  Depleted of E s s e n t i a l Amino Acids The tRNA i s o l a t i o n procedures used lead to the i s o l a t i o n of tRNA which i s nearly completely deacylated (G.M. Tener -personal communication). Thus the [ll*C] amino acid acceptance (as determined by the procedure described i n methods) for each tRNA i s a r e l a t i v e l y accurate measure of the in d i v i d u a l tRNA content i n the crude tRNA f r a c t i o n . However, i t i s necessary to express the acceptance for each amino acid r e l a t i v e to an int e r n a l standard (the acceptance for threonine) because amino acid acceptance per A 2 6 0 of d i f f e r e n t batches of crude tRNA was variable. This problem has also been encountered by other workers (3 04).. In most growth experiments, the ra t i o s of the acceptances of most amino acids to the acceptance of threonine were constant. For t h i s reason threonine was chosen as an in t e r n a l standard. To confirm the r e p r o d u c i b i l i t y of the method crude tRNA was i s o l a t e d from E. c o l i B grown i n f i v e batches under c a r e f u l l y regulated conditions (37°, no i n h i b i t o r ) . I t was found that the r a t i o s of acceptance of valine to acceptance of threonine a l l f e l l within the range 1.26 to 1.32. 48. Table 2 shows the results of amino acid acceptance measure-ment of crude tRNA from E. c o l i grown i n the presence of amino acid analogues. The tRNA from E. • c o l i B grown i n the presence of 100 Ug/ml of the tyrosine analogue, tyrosine phosphonate, had a tyrosine to threonine acceptance r a t i o of 0.65 which i s i d e n t i c a l to that of control untreated c e l l s . Growth i n the presence of 15 ug/ml of the phenylalanine analogue, p-fluoro-phenylalanine, resulted i n a crude tRNA preparation having a phenylalanine to threonine acceptance r a t i o of 0.73. This value i s , within experimental error, the same r a t i o as that of the control. E. c o l i NF162 c e l l s grown i n the presence of the valine analogue, a-amino-n-butyrate (400 ug/ml) yielded tRNA with a valine to threonine acceptance r a t i o of 1.22. The same value was obtained from the tRNA of untreated NF162 c e l l s . Table 3 shows the res u l t s of amino acid acceptance measurements of crude tRNA of E. c o l i NF162 c e l l s depleted of an esse n t i a l amino acid. As can be seen from the table, tRNA from c e l l s depleted of arginine had an arginine to threonine acceptance r a t i o of 1.94 compared to a value of 2.02 from nonstarved c e l l s . This s l i g h t difference f a l l s within experimental error. When c e l l s were depleted of methionine, the tRNA had a methionine to threo-nine acceptance r a t i o of 1.30. An i d e n t i c a l value i s obtained with nonstarved c e l l s . Based upon the y i e l d of tRNA per gram of c e l l s i s o l a t e d , i t i s calculated that approximately 3/4 of the t o t a l tRNA of the amino acid starved c e l l s was synthesized a f t e r the onset of the starvation conditions. Thus i n f i v e c l e a r l y defined experiments which give s p e c i f i c decreased leve l s of Table 2. Amino a c i d acceptance of crude tRNA from E.. c o l i grown i n the presence of amino a c i d analogues E. c o l i ' A d d i t i o n s t r a i n , Acceptance Acceptance Acceptance Acceptance Acceptance Picomoles Growth [ i - c j y a i ' ^ " c l T y r ' ["c]Phe •' [ ^ C l P r o ' [ x l ,C]Ser ' r ^ C l T h r Rate dph Acceptance Acceptance Acceptance Acceptance Acceptance Acceptance [ 1 H C ] T h r [ 1 H C ] T h r [ 1 H C ] T h r [ l 4 C ] T h r ['"ClThr per A 2 6 o u n i t B Control 1 .0 1.28 B Tyrosine Phosphonate 0.58 1.42 B p-fluorophenyl-alanine 0.58 1.26 NF162 C o n t r o l 1.09 1.22 NF162 O-methyl-DL-threonine 0.92 1.07 NF162 a-amino-n-butyrate 0.18 1.22 0.65 0.65 0.63 0.80 0.60 0.68 0.70 0.68 0.73 0.69 0.65 0.72 1.56 1.71 1.70 1.66 1.49 1.50 1.19 1.33 1.18 1.21 1.03 1.23 65 52 65 55 76 69 Table 3. Amino a c i d acceptance of crude tRNA of E. c o l i NF162 c e l l s depleted of an e s s e n t i a l amino a c i d ( Growth Growth Rate dph Acceptance [lhC]Arg Acceptance H1 "c]Met Acceptance t 1"c]Phe Acceptance [ 1"C]Tyr Acceptance f 1 " c ] V a l Acceptance [ ^ C l S e r Acceptance t 1 " c ] P r o Picomoles t 1 - C ] T h r Condition Acceptance t 1 4 C ] T h r Acceptance t 1 " c l T h r Acceptance [ l"c]Thr Acceptance t 1 "•cjThr Acceptance t 1 "clThr Acceptance [ l " c l T h r Acceptance t 1 " c l T h r Acceptance per A 2 6 o u n i t C ontrol 162 1.09 2.02 1.30 0.69 0.80 1.22 1.21 1.68 55 -Arg 0.19 1.94 0.72 0.61 1.48 1.05 1.38 61 -Met 0.29 2.25 1.30 0.65 1.15 1.51 38 o aminoacyl-tRNAs no e f f e c t was observed on the amino acid accep-tance and hence tRNA l e v e l of those s p e c i f i c tRNAs. These re-su l t s strongly argue against any r o l e for aminoacyl-tRNA l e v e l s i n the control of tRNA biosynthesis. Another experiment i n which s p e c i f i c changes i n t o t a l amino acid acceptance might be observed, but for which data have not been presented, i s that i n which E. c o l i NF162 c e l l s were grown i n the presence of the isoleucine analogue O-methyl-DL-threonine (75 ug/ml) . Rapid saturation of the isoleucine tRNA with [ 1 i*C] isoleucine was not achieved. However, i n a l l the experiments performed, the isoleucine to threonine acceptance r a t i o was less with tRNA from the analogue treated c e l l s than i n the control. I t i s possible that undermodified isoleucine tRNAs from the analogue treated c e l l s were incompletely detected because they are only slowly aminoacylated (246). Although s p e c i f i c tRNA changes were not observed, that i s changes i n tRNA species corresponding to the analogue or starva-t i o n condition used, non-specific changes i n amino acid acceptanci r a t i o s were seen (tables 2 and 3). An 11% increase i n the valine to threonine acceptance r a t i o , a 12% increase i n the serine to threonine acceptance r a t i o and a 10% increase i n the proline to threonine acceptance r a t i o was observed i n tRNA from E. c o l i B c e l l s grown i n the presence of the tyrosine phosphonate. With t h i s tRNA, no changes were observed i n the phenylalanine to threo-nine or tyrosine to threonine acceptance r a t i o s . I t i s i n t e r e s t -ing that the three amino acid acceptance r a t i o s which did change, a l l changed the same amount (approximately 11%). With tRNA from 52. p-fluorophenylalanine treated c e l l s , no changes i n acceptance r a t i o s were observed for those tRNAs accepting valin e , tyrosine, phenylalanine and serine. The proli n e to threonine acceptance r a t i o increased 9%. tRNA from NF162 c e l l s grown i n the presence of O-methyl-DL-threonine showed decreases i n the acceptance r a t i o s for valine (-12%), tyrosine (-25%), proline (-10%), and serine (-15%) but no change i n the r a t i o f o r phenylalanine accept-ance. These l a s t r e s u l t s , coupled with the threonine acceptance Thr Phe " per A 2 6 O / suggests that i n fa c t tRNA and tRNA l e v e l s i n -creased r e l a t i v e to most others i n O-methyl-DL-threonine treated c e l l s . Oddly, the tyrosine to threonine acceptance r a t i o i s decreased i n the tRNA from both O-methyl-DL-threonine treated c e l l s and a-amino-n-butyrate treated c e l l s to e s s e n t i a l l y the value obtained with tRNA from E. c o l i B c e l l s . tRNA from E. c o l i NF161 (Arg Met r e l + ) has the same r a t i o f o r tyrosine (0.8 0) under control growth conditions as does that from E. c o l i NF162. a-Amino-n-butyrate treated E. c o l i NF162 c e l l s had tRNA which showed no s i g n i f i c a n t changes i n acceptance r a t i o s for those tRNAs accepting valin e , phenylalanine and serine but showed a 15% decrease i n tyrosine and a 10% decrease i n the proline accept-ance r a t i o s . tRNA from arginine depleted E. c o l i NF162 show n e g l i g i b l e changes i n arginine and phenylalanine acceptance r a t i o s , a 21% increase i n the valine acceptance and decreases of 13% for serine, 18% for proli n e and 24% for tyrosine accept-ance r a t i o s . tRNA from methionine depleted E. c o l i NF162 showed an 11% increase i n arginine acceptance, n e g l i g i b l e changes i n the valin e and methionine acceptances and 10% and 19% decreases respectively i n proline and tyrosine acceptance r a t i o s . 53. In addition, amino acid acceptance r a t i o s were determined i n several instances which have not been included i n the tables. No change i n the glycine acceptance was observed i n tRNA from tyrosine phosphonate treated c e l l s r e l a t i v e to the c o n t r o l . This tRNA did show an 11% decrease i n alanine acceptance. With tRNA from p-fluorophenylalanine treated c e l l s , an 18% increase i n the alanine acceptance was observed but there was no difference i n the arginine acceptance r e l a t i v e to the c o n t r o l . No change i n the arginine acceptance was observed i n tRNA from a-amino-n-butyrate treated c e l l s . Since great care was taken to ensure rapid saturation of the tRNA acceptors, to normalize for variations of acceptance for d i f f e r e n t [ 1 ^ C]amino acid mixes and to do most acceptance r a t i o s at l e a s t i n quadruplicate, the acceptance r a t i o s expressed here are r e l a t i v e l y accurate measurements of the tRNA populations from c e l l s grown under the various conditions described. One can conclude that the acceptance data are not random but are char-a c t e r i s t i c of the p a r t i c u l a r growth condition. Interpretation of the data of tables 2 and 3 i s very d i f -f i c u l t . I t i s obvious that tRNA populations are not s t r i c t l y dependent upon the growth rate. I t i s i n t e r e s t i n g that growth of E. c o l i B i n the presence of the tyrosine phosphonate seems to divide the tRNA population into two groups; a group consisting of those tRNAs which accept threonine, glycine, tyrosine and phenylalanine which stay "constant" and a group consisting of those tRNAs which accept valine, serine, and proline which a l l "increase" by 10 to 12%. However t h i s larger grouping may not 54. be s i g n i f i c a n t s i n c e i t d o e s n o t h o l d u n d e r a l l o t h e r g r o w t h c o n d i t i o n s . N e v e r t h e l e s s t h e amounts o f tRNA w h i c h a c c e p t t h r e o -n i n e a n d p h e n y l a l a n i n e a r e a l m o s t p a r a l l e l i n t h e f i v e e x p e r i -m e n t a l e x a m p l e s f o r w h i c h s u f f i c i e n t i n f o r m a t i o n i s a v a i l a b l e . The d a t a a r g u e a g a i n s t t h e c e l l m a i n t a i n i n g s p e c i f i c r a t i o s o f tRNA p o p u l a t i o n s a l t h o u g h i t i s a c k n o w l e d g e d t h a t l e s s t h a n h a l f o f t h e tRNA a c c e p t a n c e r a t i o s h a v e b e e n m e a s u r e d f r o m e a c h g r o w t h c o n d i t i o n . . C o r r e l a t i o n s m i g h t become a p p a r e n t i f more a c c e p t -a n c e d a t a w e r e a v a i l a b l e . S i n c e r e c e n t w o r k (31,35) h a s shown t h a t i n E. c o l i tRNA p r e c u r s o r s a r e c o n s i d e r a b l y l a r g e r t h a n m a t u r e tRNA m o l e c u l e s a n d t h a t a s i n g l e t r a n s c r i p t may c o n t a i n p r e c u r s o r s f o r t RNAs c a p a b l e o f a c c e p t i n g s e v e r a l d i f f e r e n t a m ino a c i d s , i t i s p r o b a b l y more i m p o r t a n t t o m e a s u r e t h e l e v e l s o f i n d i v i d u a l tRNA i s o a c c e p t o r s p e c i e s . M a j o r c h a n g e s i n t h e l e v e l s o f m i n o r i s o a c c e p t o r s c o u l d r e s u l t i n o n l y s m a l l c h a n g e s i n t h e t o t a l a c c e p t a n c e f o r a p a r t i c u l a r a m ino a c i d a n d w o u l d n o t be d e t e c t e d b y t h e m e t h o d s r e p o r t e d h e r e . T h u s , i t i s i m p o r t a n t t o e s t a b l i s h i f t h e 10 t o 25% c h a n g e s i n a c c e p t a n c e r a t i o s o b s e r v e d i n t a b l e s 2 a n d 3 a r e t h e r e s u l t o f c h a n g e s i n t h e l e v e l s o f o n l y c e r t a i n i s o a c c e p t o r s o r i f a l l i s o a c c e p t o r t RNAs f o r a p a r t i c u l a r a m i n o a c i d a r e e q u a l l y a f f e c t e d . To o b t a i n t h i s d a t a t h e t R N A s w e r e f r a c t i o n a t e d o n RPC-5 a n i o n e x c h a n g e c o l u m n s . RPC-5 C h r o m a t o g r a p h y R a d i o a c t i v e l y l a b e l l e d a m i n o a c y l - t R N A s w e r e p r e p a r e d a n d f r a c t i o n a t e d o n RPC-5 c o l u m n s a s d e s c r i b e d i n M e t h o d s . The p r o -c e d u r e , c o l u m n a n d b u f f e r s y s t e m s a r e h e r e a f t e r r e f e r r e d t o a s 55. standard procedures, standard a n a l y t i c a l column and standard buffer systems. In a l l the p r o f i l e s shown the r a d i o a c t i v i t y of every f r a c t i o n was measured. Background due to deacylation was subtracted before the data were plotted. A l l p r o f i l e s were run at l e a s t twice to check on the r e p r o d u c i b i l i t y of charging and column f r a c t i o n a t i o n . The s l i g h t v a r i a b i l i t y noted i s usually due to deterioration i n resolving power of the column a f t e r a large number of experiments such that poorly resolved peaks over-lap giving a distorted picture of the amount of p a r t i c u l a r i s o -acceptors actually present. The recovery of radioactive l a b e l i n the gradient varies somewhat (70-90%). This depends on several factors including the amount of free amino acid c a r r i e d through from the preliminary chromatography of the l a b e l l e d aminoacyl-tRNA on DEAE-cellulose and the extent of deacylation during handling, freezing, thawing, etc. Several s i g n i f i c a n t changes i n the valine to threonine acceptance r a t i o s were noted previously. Figs. 1-10 show the Vcl 1 resu l t s of chromatography on RPC-5 of [ 3H]Val-tRNA from E. c o l i grown i n the presence of amino acid analogues or i n the absence of an e s s e n t i a l amino acid. Figs. 1 and 5 are the controls for [ 3H]Val-tRNA V a l from E. c o l i B and E. c o l i NF162 respectively. Both p r o f i l e s show the presence of the major valine tRNA i s o -acceptors 1, 2a and 2b and the presence of one or more very minor valine accepting tRNA species. E. c o l i B contains an additional Val valine isoacceptor, here aft e r c a l l e d tRNA^ , which elutes early on RPC-5 and which i s present i n s i g n i f i c a n t but somewhat v a r i -able amounts i n a l l the control samples examined. F i g . 2 shows Figure 1. The RPC-5 p r o f i l e of [ 3H]Val-tRNA (B). In t h i s experiment, 7.8 A 2 6 0 units of crude tRNA of E. c o l i B grown under controlled conditions at 37°C as described i n Methods and con-taining 4.6 x IO1* cpm [ 3H]Val (4% counting eff i c i e n c y ) were run on a standard RPC-5 column and eluted with the standard buffers within a l i n e a r NaCl gradient of 0.45 to 0.65 M. Approximately 90% of the [3H] applied to the column was recovered i n the gradient. The s p e c i f i c a c t i v i t y of the [3H] used i n the amino-acylation reaction was 800 uCi/umole. Figure 2. The RPC-5 p r o f i l e of [ 3H]Val-tRNA V a l (B-tyrosine phosphonate). In t h i s experiment 5.0 A 26o units of crude tRNA of E. c o l i B grown i n the presence of the tyrosine phosphonate and containing 7.6 x 1011 cpm [ 3H]Val (10% counting eff i c i e n c y ) were run as described i n F i g . 5. Approximately 85% of the ap-p l i e d r a d i o a c t i v i t y was recovered i n the gradient. The s p e c i f i c a c t i v i t y of the [ 3H]valine used i n the aminoacylation reaction was 1330 uCi/umole. 58. the [ 3 H ] V a l - t R N A V a l p r o f i l e of crude tRNA o f E. c o l i B grown i n the presence of t y r o s i n e phosphonate. An attempt has been made to q u a n t i t a t e the amounts o f i n d i v i d u a l tRNA i s o a c c e p t o r s by measuring peak areas. T h i s i s d i f f i c u l t , and hence s u b j e c t t o e r r o r , where i n d i v i d u a l tRNA i s o a c c e p t o r s are not completely r e s o l v e d . I t i s c l e a r from such measurements t h a t i n F i g . 2 there i s approximately a 100% i n c r e a s e i n the amount o f V a l - t R N A ^ a x i n c e l l s grown i n the presence o f the t y r o s i n e phosphonate. The percentage o f the t o t a l v a l i n e acceptance f o r the i s o a c c e p t o r s 2a and 2b i s s i m i l a r t o t h a t of the c o n t r o l w h i l e the amount of V a l the major i s o a c c e p t o r Val-tRNA^ i s decreased. However, the sum o f the peak areas o f the i s o a c c e p t o r s 1 and 3 i s the same i n c o n t r o l and t y r o s i n e phosphonate t r e a t e d c e l l s (72-74%). F i g . 3, showing the [ 3 H ] V a l - t R N A V a l p r o f i l e o f c e l l s grown i n the presence o f p - f l u o r o p h e n y l a l a n i n e , was analyzed i n the same V a l manner. A s i g n i f i c a n t decrease i n the amount o f Val-tRNA^ i s noted. Again the sum o f the peak areas of i s o a c c e p t o r s 3 and 1 i s e s s e n t i a l l y t h a t of the c o n t r o l (7 4% of the t o t a l v a l i n e acceptance). The peak areas of i s o a c c e p t o r s 2a and 2b are very V a l s i m i l a r t o those o f F i g . 1. F i g . 4 shows the [ H]Val-tRNA p r o f i l e o f E. c o l i B c e l l s grown i n t o the s t a t i o n a r y phase. In V a l such c e l l s Val-tRNA^ i s absent. The r e l a t i v e p e r c e n t of the V a l t o t a l acceptance o f Val-tRNA^ (73%) i s equal to the sum of the i s o a c c e p t o r s 1 and 3 of F i g . 1. That i s o a c c e p t o r s 3 and 1 can sum c o n s i s t e n t l y t o a con s t a n t v a l u e suggests t h a t both c o u l d be t r a n s c r i p t s of the same gen e ( s ) . T h i s p o s s i b i l i t y w i l l be Figure 3. The RPC-5 p r o f i l e of [ 3H]Val-tRNA (B;p-fluoro-phenylalanine). In t h i s experiment, 7.2 A 2 6 0 units of crude tRNA of E. c o l i B grown i n the presence of p-fluorophenylalanine and containing 1.87 x 10 5 cpm [ 3H]valine (10% counting e f f i c -iency) were run on RPC-5 as described i n F i g . 5. Approximately 83% of the applied r a d i o a c t i v i t y was recovered i n the gradient. The s p e c i f i c a c t i v i t y of the [ 3H]valine used i n the amino-acylation reaction was 1330 uCi/umole. Figure 4. The RPC-5 el u t i o n p r o f i l e of [ 3H]Val-tRNA V a l(B -stationary s t a t e ) . In t h i s experiment 6.8 A 26o units of crude tRNA of E. c o l i B grown into the stationary state and contain-ing 1.5 x 10 5 cpm [ 3H]Val (10% counting e f f i c i e n c y ) were run as described i n F i g . 5. Approximately 80% of the applied radio-a c t i v i t y was recovered i n the gradient. The s p e c i f i c a c t i v i t y of the [ 3H]valine used i n the aminoacylation reaction was 1300 uCi/umole. Radioactivity cpm x 10' iv: bcctivity cpm x 10 -3 —I CP O n .0 Q Q ro" h 61. d i s c u s s e d a t l e n g t h l a t e r i n t h i s t h e s i s . The i s o a c c e p t o r V a l - t R N A ^ 1 may be a p h y s i c a l l y a l t e r e d form of V a l - t R N A ^ a l o r V a l i t may be an undermodified form of Val-tRNA^ l a c k i n g p a r t i c u -l a r m o d i f i e d n u c l e o s i d e s . I f t h i s h y p o t h e s i s i s c o r r e c t , then the 11% i n c r e a s e i n the v a l i n e t o thr e o n i n e acceptance r a t i o i n t y r o s i n e phosphonate t r e a t e d c e l l s i s accomplished without a s e l e c t i v e a l t e r a t i o n i n the r e l a t i v e t r a n s c r i p t i o n o f those genes V a l coding f o r the v a l i n e i s o a c c e p t o r s 1, 2a and 2b. I f Val-tRNA^ i s a unique gene product, i . e . t r a n s c r i b e d from a gene(s) other than those f o r the i s o a c c e p t o r s 1, 2a and 2b, then s e l e c t i v e s y n t h e s i s o f the v a l i n e i s o a c c e p t o r s 3, 2a and 2b c o u l d account f o r the i n c r e a s e d acceptance r a t i o observed i n the t y r o s i n e phosphonate t r e a t e d c e l l s . T h i s i s c o n s i d e r e d a l e s s l i k e l y p o s s i b i l i t y . As shown i n F i g . 6, E. c o l i NF162 c e l l s grown i n the presence Val of 0-methyl-DL-threonine do accumulate a s m a l l amount o f Val-tRNA^ The r e l a t i v e v a l i n e tRNA i s o a c c e p t o r d i s t r i b u t i o n i s the same as t h a t of the c o n t r o l although a decrease of 12% i n the v a l i n e to thre o n i n e acceptance r a t i o was noted. D i f f e r e n t i a l t r a n s c r i p -t i o n o f i n d i v i d u a l t R N A V a x i s o a c c e p t o r s does not account f o r the a l t e r e d amino a c i d acceptance r a t i o . F i g . 7 shows the RPC-5 V a l p r o f i l e o f [ 3H]Val-tRNA from a-amino-n-butyrate t r e a t e d E. c o l i V a l NF162 c e l l s . Val-tRNA^ i s p r e s e n t i n l a r g e amounts (accounting f o r up t o 13% o f the t o t a l v a l i n e acceptance). The v a l i n e i s o -a c c e p t o r s 1, 2a and 2b are presen t although i t i s d i f f i c u l t to estimate the r e l a t i v e q u a n t i t i e s because of the presence of two V a l novel v a l i n e i s o a c c e p t o r s 13 and 2a3 . Val-tRNA_ D accounts f o r zap 62. Figure 5. The RPC-5 e l u t i o n p r o f i l e of [ 3H]Val-tRNA V a l (NF162). In t h i s experiment 7.0 A 2 6 0 units of crude tRNA of E. c o l i NF162 grown under standard conditions and containing 1.1 x 10 5 cpm [ 3H]Val (10% counting e f f i c i e n c y ) were applied to a standard a n a l y t i c a l column and eluted with standard buffers within a l i n e a r NaCl gradient from 0.50 to 0.65 M. Approximately 95% of the applied r a d i o a c t i v i t y was recovered i n the gradient. The s p e c i f i c a c t i v i t y of the [ 3H]valine used i n the aminoacylation reaction was 1300 uCi/umole. Figure 6. The RPC-5 p r o f i l e of [ 3H]Val-tRNA V a l (NF162; O-methyl-DL-threonine). In t h i s experiment, 5.8 A 2 6 o units of crude tRNA of E. c o l i NF162 grown i n the presence of O-methyl-DL-threonine and containing 1.30 x 10 5 cpm [ 3H]Val (10% counting e f f i c i e n c y ) were run on RPC-5 as described i n F i g . 5. Approximately 86% of the applied r a d i o a c t i v i t y was recovered i n the gradient. The s p e c i f i c a c t i v i t y of the [ 3H]valine used i n the aminoacylation reaction was 1330 uCi/umole. 63. 64. F i g u r e 7. The RPC-5 p r o f i l e o f [ 3 H ] V a l - t R N A V a i (NF162; a - a m i n o -n - b u t y r a t e ) . I n t h i s e x p e r i m e n t 8.0 A 2 6 0 o f c r u d e tRNA o f E. c o l i NF162 g r o w n i n t h e p r e s e n c e o f a - a m i n o - n - b u t y r a t e a nd c o n t a i n i n g 2.20 x 1 0 5 cpm [ 3 H ] V a l ( 1 0 % c o u n t i n g e f f i c i e n c y ) w e r e r u n a s d e s c r i b e d i n F i g . 5. A p p r o x i m a t e l y 8 5% o f t h e a p p l i e d r a d i o a c t i v i t y was r e c o v e r e d i n t h e g r a d i e n t . The [ 3 H ] v a l i n e u s e d i n t h e a m i n o a c y l a t i o n r e a c t i o n h a d a s p e c i f i c a c t i v i t y o f 1300 u C i / u m o l e . F i g u r e 8. The c o c h r o m a t o g r a p h y on RPC-5 o f [ 1 "*C] V a l - t R N A (B) and [ 3 H ] V a l - t R N A V a l ( 1 6 2 - A r g ) . I n t h i s e x p e r i m e n t 10.4 A 2 6 o u n i t s o f c r u d e tRNA o f E. c o l i B c o n t a i n i n g 3.30 x 1 0 s cpm t ^ C ] V a l ( 8 0 % c o u n t i n g e f f i c i e n c y ) p l u s 6.27 A 2 6 o u n i t s o f c r u d e tRNA o f E. c o l i NF162 d e p l e t e d o f a r g i n i n e a n d c o n t a i n i n g 2.64 x 1 0 s cpm [ 3 H ] V a l ( 3 7 % c o u n t i n g e f f i c i e n c y ) w e r e r u n on RPC-5 a s d e s -c r i b e d i n F i g . 5. A p p r o x i m a t e l y 76% o f t h e a p p l i e d [ 1 "*C] a nd 7 2 % o f t h e a p p l i e d [ 3H] r a d i o a c t i v i t y was r e c o v e r e d i n t h e g r a d i e n t . The [ 3 H ] v a l i n e a n d [ 1 ^ C ] v a l i n e u s e d i n t h e a m i n o -a c y l a t i o n r e a c t i o n s h a d s p e c i f i c a c t i v i t i e s o f 13 00 u C i / u m o l e and 266 u C i / u m o l e r e s p e c t i v e l y . Radioactivity cpm x 10" a p p r o x i m a t e l y 10% o f t h e t o t a l v a l i n e a c c e p t a n c e . I t i s l i k e l y t h a t t h e n o v e l i s o a c c e p t o r s 13 and 2a3 r e p r e s e n t u n d e r m o d i f i e d f o r m s o f t h e m a j o r i s o a c c e p t o r s n o r m a l l y o b s e r v e d . S i n c e t h e i d e n t i t y o f t h e s e p e a k s a n d t h e v a l i n e i s o a c c e p t o r 3 p r e v i o u s l y d i s c u s s e d i s n o t known, i t i s n o t p o s s i b l e t o e s t i m a t e t h e r e l a -t i v e q u a n t i t i e s o f t h e t r a n s c r i p t i o n p r o d u c t s o f t h e p a r t i c u l a r V a l tRNA g e n e s . A l t h o u g h no o v e r a l l c h a n g e i n t h e v a l i n e t o t h r e o n i n e a c c e p t a n c e r a t i o was o b s e r v e d i t r e m a i n s t o be p r o v e n V a l t h a t a l t e r e d t r a n s c r i p t i o n o f p a r t i c u l a r tRNA g e n e s d i d n o t o c c u r . V a l F i g u r e s 8 t o 10 show t h e l a b e l l e d V a l - t R N A p r o f i l e s f r o m a r g i n i n e d e p l e t e d a n d m e t h i o n i n e d e p l e t e d E. c o l i NF162. Co-c h r o m a t o g r a p h y h a s b e e n u s e d i n o r d e r t h a t a c o m p a r i s o n c a n be made o f t h e v a l i n e i s o a c c e p t o r s p r e s e n t u n d e r t h e d i f f e r e n t g r o w t h c o n d i t i o n s . T h i s i s e s p e c i a l l y i m p o r t a n t b e c a u s e o f t h e c o m p l e x -i t y o f t h e i s o a c c e p t o r d i s t r i b u t i o n s o b s e r v e d . C o c h r o m a t o g r a p h y V e i l w i t h [ 1 4 C ] V a l - t R N A (B) h a s b e e n u s e d i n s t e a d o f c o c h r o m a t o -g r a p h y w i t h [ 1 ^ C ] V a l - t R N A V a l (NF162) b e c a u s e t h e f o r m e r c o n t a i n s , i n a d d i t i o n t o t h o s e v a l i n e i s o a c c e p t o r s o b s e r v e d i n E. c o l i N F 1 6 2 , V a l - t R N A ^ 3 1 w h i c h s e r v e s a s a r e f e r e n c e p e a k f o r e a r l y V a l e l u t i n g V a l - t R N A i s o a c c e p t o r s . I n f i g u r e 8, i t c a n be s e e n t h a t a r g i n i n e d e p l e t e d E. c o l i NF162 c e l l s h a v e a V a l - t R N A V a l p r o f i l e o n RPC-5 v e r y much l i k e t h a t f o r a - a m i n o - n - b u t y r a t e t r e a t e d E. c o l i B c e l l s . The m a j o r v a l i n e i s o a c c e p t o r s 1, 2a a n d 2b a r e p r e s e n t a s a r e t h e n o v e l v a l i n e i s o a c c e p t o r s 3, I B V a l a n d 2ag p r e v i o u s l y o b s e r v e d . V a l - t R N A ^ i s a g a i n p r e s e n t i n v e r y s i g n i f i c a n t q u a n t i t i e s . The amount o f 13 p r e s e n t i n t h e a r g i n i n e F i g u r e 9. The c o c h r o m a t o g r a p h y o n RPC-5 o f [ 1 ^ C ] V a l - t R N A a (B) a n d [ 3 H ] V a l - t R N A V a l ( 1 6 2 - M e t ) . I n t h i s e x p e r i m e n t 5.9 A 2 6 o u n i t s o f c r u d e tRNA o f E . c o l i B c o n t a i n i n g 1.55 x 1 0 5 cpm [ ^ C j V a l (8 0 % c o u n t i n g e f f i c i e n c y ) p l u s 7.5 A 2 6 o u n i t s o f c r u d e tRNA o f E . c o l i NF162 d e p l e t e d o f m e t h i o n i n e a n d c o n t a i n i n g 4.28 x 1 0 5 cpm [ 3 H ] V a l ( 3 7 % c o u n t i n g e f f i c i e n c y ) w e r e r u n o n RPC-5 a s d e s -c r i b e d i n F i g . 5. A p p r o x i m a t e l y 82% of. t h e a p p l i e d [ 3H] l a b e l a n d 85% o f t h e a p p l i e d [lkC] l a b e l w e r e r e c o v e r e d i n t h e g r a d i e n t . The s p e c i f i c a c t i v i t i e s o f t h e [ 3 H ] v a l i n e a n d [lhC]valine u s e d i n t h e a m i n o a c y l a t i o n r e a c t i o n w e r e 13 00 u C i / u m o l e a n d 266 u C i / u m o l e r e s p e c t i v e l y . F i g u r e 10. The c o c h r o m a t o g r a p h y o n RPC-5 o f [ 3 H ] V a l - t R N A V a l (NF162-Arg) a n d [ 1 "*C] V a l - t R N A V a l (NF162-Met) . I n t h i s e x p e r i m e n t 6.3 A 2 6 o u n i t s o f c r u d e tRNA o f m e t h i o n i n e d e p l e t e d E . c o l i NF162 c o n t a i n i n g 1.1 x 1 0 s cpm [ 1 ' * C ] V a l ( 8 0 % c o u n t i n g e f f i c i e n c y ) p l u s 9.5 A 2 6 o u n i t s o f c r u d e tRNA o f a r g i n i n e d e p l e t e d E . c o l i NF162 c o n t a i n i n g 9.1 x 1 0 s cpm [ 3 H ] V a l ( 3 7 % c o u n t i n g e f f i c i e n c y ) w e r e r u n a s d e s c r i b e d i n F i g . 5. A p p r o x i m a t e l y 82% o f t h e a p p l i e d [ 3H] a n d 96% o f t h e a p p l i e d [ l l fC] w e r e r e c o v e r e d i n t h e g r a d i e n t . The s p e c i f i c a c t i v i t i e s o f t h e [ 3H] v a l i n e a n d [ 1 **C] v a l i n e u s e d i n t h e a m i n o a c y l a t i o n r e a c t i o n was 1300 u C i / u m o l e a n d 266 u C i / u m o l e r e s p e c t i v e l y . o i H (D U •H CM e . 0 l x |JH£] 68, CM ... , . i < i 1 i 1 1 1 | 1 | 1 - < CN o „ o l CN > A z at 0 0 . CN / -• • / " 1 _ ' X ' ICO i JO S~ cy — ^ CN  — > + CO 1 i - CN - c z " 1 -1 i 1 i 1 i 1 1 1 1 o CM •2-0l x web [D^ o CO o CO o o a z O L U CO a; u tji -H CM CD z CJ I CN Z i£2_J .01 x Uicb CM CM ~ r c - o i x web depleted c e l l s i s considerably less than that observed i n a-amino-n-butyrate treated c e l l s . It i s i n t e r e s t i n g that such very d i f f e r -ent growth conditions give r i s e to the same novel valine i s o -acceptors. This argues against the novel isoacceptors being formed i n response to s p e c i f i c environmental stress i . e . valine analogue treated c e l l s do not have s p e c i f i c novel valine tRNA isoacceptors. I t i s also noted that arginine depleted E. c o l i NF162 accumulate small amounts of valine isoacceptors which elute between Val-tRNA^ 1 and Val-tRNA^ a l. Methionine depleted E. c o l i NF162 give quite a d i f f e r e n t tRNA p r o f i l e on RPC-5. A small Val amount of Val-tRNA^ i s observed. New valine isoacceptors 3 3 and 3 y are observed i n small amounts. Val-tRNA^ a l and Val-tRNA^ 1 are both present i n s i g n i f i c a n t quantities. As i n arginine de-pleted c e l l s the v a l i n e isoacceptors 1 3 and 2a3 are observed. Val I t i s d i f f i c u l t to estimate the amount of Val-tRNA- present because of the presence of the new v a l i n e isoacceptor 2aa. F i g . 10 confirms the r e s u l t s of the previous two figures and makes more obvious some of the quantitative differences i n i n d i v i d u a l valine isoacceptors. Since the i d e n t i t y of the novel valine isoacceptors i s not known i t i s not possible to discuss the re s u l t s obtained i n terms of the amount of t r a n s c r i p t i o n of par-Val t i c u l a r tRNA genes. The novel valine isoacceptors observed are i n themselves i n t e r e s t i n g . They are l i k e l y undermodified forms of the valine isoacceptors normally observed. Figures 11 and 12 show the RPC-5 p r o f i l e s of [ 3H]Ser-tRNA S e r from control and tyrosine phosphonate treated c e l l s respectively. S i g n i f i c a n t increases i n the amounts of two of the serine i s o -70. F i g u r e 1 1 . The RPC-5 p r o f i l e o f [ 1 " c ] S e r - t R N A S e r ( B ) . I n t h i s e x p e r i m e n t 6.2 A 2 6 0 u n i t s o f c r u d e tRNA o f E. c o l i B c o n t a i n i n g 1.05 x 1 0 5 cpm ( 7 6 % c o u n t i n g e f f i c i e n c y ) w e r e a p p l i e d t o a s t a n -d a r d a n a l y t i c a l c o l u m n a n d e l u t e d w i t h s t a n d a r d b u f f e r s w i t h i n a l i n e a r N a C l g r a d i e n t o f 0.45 t o 1.5 M. A p p r o x i m a t e l y 80% o f t h e a p p l i e d r a d i o a c t i v i t y was r e c o v e r e d i n t h e g r a d i e n t . The [ 1 < fC] s e r i n e u s e d i n t h e a m i n o a c y l a t i o n r e a c t i n g h a d a s p e c i f i c a c t i v i t y o f 149 u C i / u m o l e . F i g u r e 12. The RPC-5 p r o f i l e o f [ 1 * C ] S e r - t R N A S e r ( B - T y r o s i n e P h o s p h o n a t e ) . I n t h i s e x p e r i m e n t 4.0 A 2 6 o u n i t s o f c r u d e tRNA o f E. c o l i B g r o w n i n t h e p r e s e n c e o f t h e t y r o s i n e p h o s p h o n a t e and c o n t a i n i n g 6.9 x 10 1* cpm [ 1 "*C] S e r ( 7 6 % c o u n t i n g e f f i c i e n c y ) w e r e r u n a s d e s c r i b e d i n F i g . 1 1 . A p p r o x i m a t e l y 90% o f t h e a p -p l i e d r a d i o a c t i v i t y was r e c o v e r e d i n t h e g r a d i e n t . The [1'*C] s e r i n e u s e d i n t h e a m i n o a c y l a t i o n r e a c t i o n h a d a s p e c i f i c a c -t i v i t y o f 149 u C i / u m o l e . Figure 11 n i o x 3 E CL u U L.2 v O o I I i i r [UC] Ser tRNA S o r (B-Control) 71, 60 80 100 120 140 160 100 200 T U B E N O . 1-5 o z ]1-0 ^ o £ —40-5 — m 1 1 1 1 1 i i i r Figure 12 [>4C] S E R »RNA W (B-Tyrosine-Phosphonate) i o E a o y 1 O q o 100 120 140 ' 160 180 200 T U B E N O . a c c e p t o r s a r e n o t e d i n F i g . 12. The i n c r e a s e s i n t h e s e p e a k s , ( t h e 3 r d & 4 t h m a j o r s e r i n e i s o a c c e p t o r s i n o r d e r o f e l u t i o n f r o m RPC-5 i n t h e c o n t r o l e x p e r i m e n t ) f r o m a p p r o x i m a t e l y 1 5 % o f t h e t o t a l a c c e p t a n c e t o a p p r o x i m a t e l y 2 1 % o f t h e t o t a l a c c e p t -a n c e f o r t h e 3 r d m a j o r i s o a c c e p t o r , a n d f r o m a p p r o x i m a t e l y 1 0 % o f t h e t o t a l a c c e p t a n c e t o a p p r o x i m a t e l y 14% o f t h e t o t a l a c c e p t -a n c e f o r t h e 4 t h m a j o r i s o a c c e p t o r c o u l d c o n c e i v a b l y a c c o u n t f o r t h e 1 2 % i n c r e a s e i n t h e s e r i n e t o t h r e o n i n e a c c e p t a n c e r a t i o o b -s e r v e d . I t i s p o s s i b l e i n t h i s i n s t a n c e t h a t a s p e c i f i c s y n -t h e s i s o f two s e r i n e i s o a c c e p t o r s o c c u r r e d i n r e s p o n s e t o g r o w t h i n h i b i t i o n b y t y r o s i n e p h o s p h o n a t e . Why o r how s u c h a s p e c i f i c c h a n g e c o u l d h a v e o c c u r r e d i s n o t o b v i o u s . F i g u r e s 13 t o 15 show t h e [ 3 H ] S e r - t R N A S e r p r o f i l e s f o r c o n t r o l NF162 c e l l s , NF162 c e l l s t r e a t e d w i t h a - a m i n o - n - b u t y r a t e a n d NF162 c e l l s d e p l e t e d o f a r g i n i n e . F i g u r e s 13 and 14 show s u p e r i o r r e s o l u t i o n t o t h a t o f F i g u r e s 1 1 , 12 a n d 15 b e c a u s e RPC-5 o f 50 mesh s i e v e s i z e was u s e d i n t h e l a t t e r e x p e r i m e n t s w h i l e RPC-5 o f 200 mesh s i e v e s i z e was u s e d i n a l l o t h e r RPC-5 p r o f i l e s i n t h i s t h e s i s . F i g . S e r 14 shows a s l i g h t l y a l t e r e d S e r - t R N A p r o f i l e . A number o f i n d i s t i n c t s e r i n e i s o a c c e p t o r s a r e o b s e r v e d i n f r a c t i o n s 90 t o 13 0. T h e r e a p p e a r s t o be l e s s m a t e r i a l i n t h e f i n a l two m a j o r s e r i n e i s o a c c e p t o r s . The e a r l i e r e l u t i n g s p e c i e s may i n f a c t be p r e c u r s o r s o f t h e l a t e r e l u t i n g o n e s . No o v e r a l l c h a n g e i n t h e s e r i n e a c c e p t a n c e r a t i o was o b s e r v e d a s p r e v i o u s l y i n d i c a t e d . a - A m i n o - n - b u t y r a t e t r e a t m e n t c a u s e s a much more d r a s t i c a l t e r a -V a l S e r t i o n i n i s o a c c e p t o r d i s t r i b u t i o n o f V a l - t R N A t h a n S e r - t R N A The [ 3 H ] S e r - t R N A S e r ( 1 6 2 - A r g ) ( F i g . 15) p r o f i l e d i f f e r s f r o m t h a t F i g u r e 1 3 . The RPC-5 p r o f i l e o f [ 3 H ] S e r - t R N A S e r NF162. I n t h i s e x p e r i m e n t 6.7 A 26o u n i t s o f c r u d e tRNA f r o m E. c o l i NF162 c o n -t a i n i n g 4.2 x 1 0 s cpm [ 3 H ] S e r ( 1 0 % c o u n t i n g e f f i c i e n c y ) w e r e r u n as d e s c r i b e d i n F i g . 1 1 . A p p r o x i m a t e l y 90% o f t h e a p p l i e d r a d i o -a c t i v i t y was r e c o v e r e d i n t h e g r a d i e n t . The [ 3 H ] s e r i n e u s e d i n t h e a m i n o a c y l a t i o n r e a c t i o n h a d a s p e c i f i c a c t i v i t y o f 3370 u C i / u m o l e . F i g u r e 14. The RPC-5 p r o f i l e o f [ 3 H ] S e r - t R N A S e r (NF162; a - a m i n o -n - b u t y r a t e ) . I n t h i s e x p e r i m e n t 10.0 A 2 6 0 u n i t s o f c r u d e tRNA f r o m E. c o l i NF162 g r o w n i n t h e p r e s e n c e o f t h e a n a l o g u e a - a m i n o -n - b u t y r a t e a n d c o n t a i n i n g 4.5 x 1 0 5 cpm [ 3 H ] S e r ( 1 0 % c o u n t i n g e f f i c i e n c y ) w e r e r u n a s d e s c r i b e d i n F i g . 1 1 . A p p r o x i m a t e l y 90% o f t h e a p p l i e d r a d i o a c t i v i t y was r e c o v e r e d i n t h e g r a d i e n t . The [ 3 H ] s e r i n e u s e d i n t h e a m i n o a c y l a t i o n r e a c t i o n h a d a s p e c i f i c a c t i v i t y o f 3370 u C i / u m o l e . F i g u r e 13 20 40 60 80 100 120 140 160 180 F i g u r e 14 T U B E N O . 200 20 o i E a u X o 1 0 u D O O n 1 1 n r 1 1 r [3H] SertRNA S e r (NF-162; o-NH2-n-butyric acid) 20 40 60 80 100 120 140 T U B E N O . 160 180 75. F i g u r e 15. The RPC-5 p r o f i l e o f [ 3 H ] S e r - t R N A S e r ( N F 1 6 2 - A r g ) . I n t h i s e x p e r i m e n t 9.0 A 2 6 o u n i t s o f c r u d e tRNA o f E. c o l i NF162 d e p r i v e d o f a r g i n i n e , c o n t a i n i n g 4.0 x 1 0 5 cpm [ 3 H ] S e r ( 1 0 % c o u n t i n g e f f i c i e n c y ) w e r e r u n a s d e s c r i b e d i n F i g . 1 1 . A p p r o x i m a t e l y 90% o f t h e a p p l i e d r a d i o a c t i v i t y was r e c o v e r e d i n t h e g r a d i e n t . The [ 3 H ] s e r i n e u s e d i n t h e a m i n o a c y l a t i o n r e a c t i o n h a d a s p e c i f i c a c t i v i t y o f 337 0 u C i / u m o l e . o f t h e c o n t r o l F i g . 1 3 , p a r t i c u l a r l y i n t h e r e g i o n f r a c t i o n 100 t o f r a c t i o n 120. T h e r e a p p e a r s t o be a n i n c r e a s e o f m a t e r i a l i n t h i s l a t t e r r e g i o n a n d a d e c r e a s e i n t h e s i z e o f t h e l a t e r e l u t -i n g p e a k s . The r e g i o n c o n t a i n i n g t h e two m a j o r i s o a c c e p t o r s i s r e d u c e d t o a p p r o x i m a t e l y 50% o f t h e t o t a l s e r i n e a c c e p t a n c e f r o m t h e c o n t r o l v a l u e o f a p p r o x i m a t e l y 66%. I f we assume t h a t t h e tRNA i n t h e r e g i o n o f f r a c t i o n s 100 t o 120 i s t r a n s c r i b e d f r o m t h e same g e n e s a s t h e f i n a l e l u t i n g s e r i n e i s o a c c e p t o r s t h e n t h e 1 3 % d e c r e a s e i n t h e s e r i n e t o t h r e o n i n e a c c e p t a n c e r a t i o i s e a s i l y a c c o u n t e d f o r by a s p e c i f i c d e c r e a s e i n t h e s y n t h e s i s o f t h e m a j o r s e r i n e i s o a c c e p t o r o f f r a c t i o n s 65 t o 90 o f F i g . 15. T h i s a r g u m e n t i s t e n u o u s and s e r v e s t o p o i n t o u t t h e n e e d t o i d e n -t i f y t h e e x a c t g e n e o r i g i n o f a l l t h e tRNA i s o a c c e p t o r s f o r a p a r t i c u l a r a m i n o a c i d . F i g . 16 shows t h e [ 3 H ] L e u - t R N A L e u (NF162) p r o f i l e o n RPC-5. T h i s p r o f i l e i s e s s e n t i a l l y t h e same a s t h a t o f t h e [ l l t C ] L e u -t R N A L e u ( B - c o n t r o l ) u s e d f o r c o c h r o m a t o g r a p h y i n F i g u r e s 17 and 18. F i g u r e s 17 a n d 18 show t h e c o c h r o m a t o g r a p h y o f [ l l f C ] L e u -t R N A L e u ( B - c o n t r o l ) and [ 3 H ] L e u - t R N A L e u o f a - a m i n o - n - b u t y r a t e t r e a t e d NF162 c e l l s a n d a r g i n i n e d e p l e t e d NF162 c e l l s r e s p e c -t i v e l y . T h e s e p r o f i l e s a r e i n c l u d e d t o d e m o n s t r a t e t h e e x t e n s i v e f o r m a t i o n o f n o v e l l e u c i n e i s o a c c e p t o r s u n d e r t h e s e g r o w t h c o n -d i t i o n s . O b v i o u s l y d i f f e r e n t t RNAs h a v e a d i f f e r e n t p o t e n t i a l t o f o r m n o v e l i s o a c c e p t o r s i n a p a r t i c u l a r g r o w t h c o n d i t i o n . The l i t e r a t u r e c o n t a i n s s e v e r a l p u b l i c a t i o n s r e l a t e d t o t h i s w o r k . Wong e t a l . (190) s u g g e s t t h a t when g r o w t h o f e i t h e r a l e u c i n e o r t r y p t o p h a n a u x o t r o p h was l i m i t e d by t h e s u p p l y o f 100 120 140 160 180 200 T U B E N O . Figure 16. The RPC-5 eluti o n p r o f i l e of [ 1^C]Leu-tRNA L e u (NF162). In t h i s experiment 6.4 A 2 6 0 units of crude tRNA of E. c o l i NF162 containing 2.9 x 10 5 cpm [ 1 "*C] Leu (80% counting eff i c i e n c y ) were run on a standard a n a l y t i c a l column and eluted with standard buffers within a l i n e a r NaCl gradient of 0.50 to 0.95 M. Approximately 71% of the applied r a d i o a c t i v i t y was recovered i n the gradient. The s p e c i f i c a c t i v i t y of the [ 1 ! tC] leucine used i n the aminoacylation reaction was 294 uCi/umole. 78. 60 80 100 120 140 160 180 T U B E N O . Figure 17. The cochromatography of [ 3H]Leu-tRNA L e u (NF162: a-amino-n-butyrate) and [ 1 kC] Leu-tRNA L e u (B) . In t h i s experi-ment, crude tRNA of E. c o l i NF162 grown i n the presence of the valine analogue a-amino-n-butyrate and containing 1.3 5 x 10 5 cpm [ 3H]Leu (37% counting eff i c i e n c y ) and crude tRNA of E. c o l i B containing 9.5 x 10 k cpm [ 1^C]Leu (8 0% counting eff i c i e n c y ) were run as described i n F i g . 16. Approximately 85% of the applied [3H] and 82% of the applied [ 1 4C] were recovered i n the gradient. The s p e c i f i c a c t i v i t i e s of the [ 3H].leucine and [ 1 4C] leucine used i n the aminoacylation reactions were 5,000 uCi/umole and 294 uCi/umole respectively. The leucine to threonine accept-ance r a t i o was 1.60 for the crude tRNA of (NF162; a-amino-n-butyrate) . F i g u r e 18. The cochromatography of [ 3H]Leu-tRNA (NF162:-Arg) and [ 1 "*C] L e u - t RNA L e u (B) . In t h i s experiment, crude tRNA of E. c o l i NF162 d e p l e t e d of a r g i n i n e and c o n t a i n i n g 1.33 x 10 5 cpm T 3H]Leu (37% c o u n t i n g e f f i c i e n c y ) and crude tRNA of E. c o l i B c o n t a i n i n g 9.5 x 10 k cpm [ ^ C j L e u (80% cou n t i n g e f f i c i e n c y ) were run as d e s c r i b e d i n F i g . 16. Approximately 8 5% of the a p p l i e d [ 3H] and 84% of the a p p l i e d [ 1 < fC] was recovered i n the g r a d i e n t . The s p e c i f i c a c t i v i t i e s o f the [ 3 H ] l e u c i n e and [ 1 h C ] l e u c i n e used i n the a m i n o a c y l a t i o n r e a c t i o n s were 5,000 uCi/umole and 294 uCi/umole r e s p e c t i v e l y . The [ 1 ^ C ] l e u c i n e acceptance t o [lkC] t h r e o n i n e acceptance r a t i o was 1.8 5 f o r the crude tRNA of (NF162-Arg) . 80. amino acid no s p e c i f i c changes were observed i n the tRNA leve l s corresponding to the l i m i t i n g amino acid. The design of t h e i r experiments was such that i t i s possible that s p e c i f i c tRNA control might not be observed under the experimental conditions they used and thus we sought to confirm or refute t h e i r observa-tions i n a more c l e a r l y defined system. Our r e s u l t s confirm the r e s u l t s previously reported i n that we likewise observed no s p e c i f i c changes i n tRNA l e v e l s when E. c o l i NF162 c e l l s were depleted of arginine or methionine. We also attempted to extend the previous work by analyzing i n d i v i d u a l isoacceptor d i s t r i b u -t i o n . Large changes i n the phenylalanine to l y s i n e acceptance r a t i o have been reported i n E. c o l i ( r e l Arg ) starved of arg-inine (270). Differences i n response of stringent and relaxed c e l l s to arginine deprivation were also noted. In an i n t e r e s t i n g experiment Wong et a l . (190) plotted tRNA content versus growth rate over a wide range of growth rates. Straight l i n e s of d i f f e r e n t slopes were obtained for tRNA L e u, H e Trv • • tRNA , and tRNA •*. Since the slopes were not i d e n t i c a l , t h i s suggests that d i f f e r e n t growth rates have quite d i f f e r e n t tRNA populations (one tRNA r e l a t i v e to another). This argues for s p e c i f i c control of tRNA biosynthesis. I t also poses the pos-s i b i l i t y that i n amino acid analogue treated c e l l s , the tRNA population i s more c h a r a c t e r i s t i c of the growth rate than the growth i n h i b i t o r . This, however, i s disproved by the fact that tyrosine phosphonate treated c e l l s and p-fluorophenylalanine treated c e l l s , having the same growth rate, show very s i g n i f i c a n t differences i n t h e i r tRNA populations. The i d e n t i c a l 10% increase 81. P re-i n the tRNA content i n each experiment could be because of the i d e n t i c a l change i n growth rate. More than one tRNA control mechanism may be operative under any p a r t i c u l a r set of growth conditions. Skjold et a l . (298) observe no changes i n r e l a t i v e i s o -acceptor d i s t r i b u t i o n for a l l the tRNAs examined for tRNA from slow and fa s t growing E. c o l i . They postulated that isoaccep-tors are regulated as a group. Our r e s u l t s suggest the same idea. S i g n i f i c a n t differences were observed i n the serine to valine acceptance r a t i o of c e l l s grown on acetate as a carbon source (0.55) compared to c e l l s grown on nutrient broth (1.17). Numer-ous other changes i n acceptance r a t i o s were also observed suggest-ing some form of tRNA control as a function of growth rate. Changes i n amino acid acceptance r a t i o s and altered amounts of p a r t i c u l a r tRNA isoacceptors can r e s u l t from a d i f f e r e n t i a l rate of nuclease digestion of p a r t i c u l a r tRNAs. I t has been re-ported that mutations which d e s t a b i l i z e tRNA structure rendering those tRNAs more susceptible to nuclease attack (268,269) or mutations which r e s u l t i n slower processing of tRNA precursors (268,269) allowing for nonspecific nuclease cleavage, r e s u l t i n correspondingly decreased tRNA l e v e l s . I t i s conceivable that tRNAs d e f i c i e n t i n c e r t a i n modified nucleosides have an altered s u s c e p t i b i l i t y to nuclease action. Hence tRNA l e v e l s and i s o -acceptor d i s t r i b u t i o n can change without a s i g n i f i c a n t change i n the actual rate of gene t r a n s c r i p t i o n . Uncleaved precursor tRNAs lacking modified nucleosides are not processed more slowly than those having modified nucleosides (27 5). An inte r e s t i n g 82. h y p o t h e s i s (274) h a s b e e n p u t f o r w a r d s u g g e s t i n g t h a t t h e i n -c r e a s e d 2 * - 0 - m e t h y l a t i o n o b s e r v e d i n B a c i l l u s s t e a r o t h e r m o p h i l u s g r own a t h i g h t e m p e r a t u r e r e s u l t s i n a s t a b i l i z a t i o n o f t h e t R N A s a g a i n s t n u c l e a s e a t t a c k when t h e tRNAs h a v e a more o p e n s t r u c -t u r e . I f t h e h y p o t h e s i s i s c o r r e c t , d i f f e r e n t i a l 2 ' - 0 - m e t h y l a -t i o n c o u l d s i g n i f i c a n t l y a l t e r i n d i v i d u a l a m ino a c i d a c c e p t a n c e and r e l a t i v e i s o a c c e p t o r d i s t r i b u t i o n . I t i s a l s o p o s s i b l e t h a t t R N A s may u n d e r g o s p e c i f i c l o s s o f t h e i r CCA t e r m i n i . T h i s p o s s i b i l i t y h a s n o t b e e n e x a m i n e d i n t h e s e e x p e r i m e n t s . T u r n o v e r o f t h e CCA e n d h a s b e e n shown (61) t o v i r t u a l l y c e a s e i n a m i n o a c i d s t a r v e d E. c o l i . N o v e l tRNA i s o a c c e p t o r s h a v e b e e n r e p o r t e d i n E. c o l i g r o w n u n d e r a w i d e v a r i e t y o f a d v e r s e g r o w t h c o n d i t i o n s . S t a r -v a t i o n o f a l e u c i n e a u x o t r o p h f o r l e u c i n e r e s u l t e d i n t h e f o r m -a t i o n o f n o v e l tRNA i s o a c c e p t o r s f o r l e u c i n e , h i s t i d i n e , a r g i n i n e , v a l i n e a n d p h e n y l a l a n i n e ( 2 9 2 ) . S t a r v a t i o n o f a h i s t i d i n e a u x o -t r o p h f o r h i s t i d i n e d i d n o t y i e l d n o v e l i s o a c c e p t o r s f o r l e u c i n e o r a r g i n i n e a l t h o u g h s t a r v a t i o n o f an a r g i n i n e a u x o t r o p h f o r a r g i n i n e d i d ( 2 9 2 ) . I n o u r h a n d s , s t a r v a t i o n o f a n a r g i n i n e a u x o t r o p h f o r a r g i n i n e r e s u l t e d i n t h e f o r m a t i o n o f n o v e l i s o -a c c e p t o r s f o r v a l i n e a n d l e u c i n e a nd c h a n g e s i n i s o a c c e p t o r d i s t r i b u t i o n f o r s e r i n e . S t a r v a t i o n o f a p h e n y l a l a n i n e a u x o t r o p h f o r p h e n y l a l a n i n e r e s u l t e d i n n o v e l i s o a c c e p t o r f o r m a t i o n f o r Phe tRNA ( 1 7 3 ) . S t a r v a t i o n o f a r e l a x e d c o n t r o l m e t h i o n i n e a u x o -t r o p h f o r m e t h i o n i n e h a s b e e n shown t o be a p a r t i c u l a r l y e f f e c -t i v e means o f o b t a i n i n g u n d e r m o d i f i e d tRNA. M e t h y l d e f i c i e n t tRNA i s o a c c e p t o r s f o r p h e n y l a l a n i n e (18 6 , 1 8 8 , 2 9 3 , 2 9 4 , 2 9 5 ) , 83. leucine (296), v a l i n e (179,180) and methionine (246) have been studied. Growth of E. c o l i i n the presence of chloramphenicol has been used as a means of obtaining undermodified tRNAs for phenyl-alanine (172,173,175,291), leucine (172,173), and methionine, Phe arginine, l y s i n e , isoleucine and serine (173). Novel tRNA isoacceptors have been observed i n E. c o l i grown on a low phos-phate medium at a slow growth rate (171). E. c o l i grown i n a medium with less than 10 7 M iron or grown under l i m i t i n g aeration Phe i n an aged medium show novel tRNA isoacceptors (170). I t has been shown that the l a t t e r novel isoacceptors lack ms 2i 6A • K Phe and % A (176). The novel tRNA isoacceptors of low phosphate media are thought to lack these same modifications (171) as are Phe the tRNA isoacceptors of chloramphenicol treated c e l l s (175). Chloramphenicol treated c e l l s have been shown also to be d e f i c i e n t i n the minor nucleosides hU,, s^U, m2A and m7G, m1G (173,290,291). A pseudouridine deficiency i n tRNA was noted i n E. c o l i treated with 2 - t h i o u r a c i l (297). tRNA of methionine starved methionine auxotrophs i s methyl d e f i c i e n t (179,180,186,188,293-296). Other nucleoside modifications are reduced as well (173) and not a l l methyl modified nucleosides are equally methyl d e f i c i e n t (173,185). Most l i k e l y the novel isoacceptors observed i n the experiments described i n t h i s thesis lack modified nucleosides. I t i s i n t e r -esting that amino ac i d analogues give r i s e to many of the same novel isoacceptor tRNAs observed under conditions of amino acid starvation. To c l a r i f y the l i t e r a t u r e i t would be h e l p f u l i f attempts were made to r e l a t e the various novel isoacceptors from the d i f f e r e n t growth conditions. 84. In several instances i t has been shown that undermodified tRNAs can have d i f f e r e n t functional properties (137 and references c i t e d therein). In one instance i t has been suggested that undermethylated tRNA has a reduced capacity to accept amino acids (247). Other laboratories have suggested that methyl d e f i c i e n t tRNA accepts amino acids although at a somewhat reduced rate, the f i n a l l e v e l of acylation obtained being the same (246). In the experiments described i n t h i s thesis s u f f i c i e n t amino-acyl-tRNA synthetase was used to ensure rapid saturation of the acceptors. Acceptance was measured at 5, 10 at 20 minutes to demonstrate complete acylation. Those undermodified forms capable of accepting amino acids were completely acylated. However, undermodified tRNAs incapable of any aminoacylation obviously would not be detected. It i s not possible to predict the precise nature of the modified nucleoside deficiency based on e l i i t i o n p o s ition on RPC. Several tRNAs lacking elute at higher s a l t concentration on RPC than the f u l l y modified forms (310,249). Undermethylated tRNAs have been shown to elute both e a r l i e r (179,24 6) and l a t e r (183) than the f u l l y mature tRNA. tRNAs lacking hydrophobic modified nucleosides such as i 6 A and m s 2 £ 6 A elute at lower s a l t concentrations (170,175). tRNA from E. c o l i grown i n the presence of chloramphenicol i s d e f i c i e n t i n ms 2i 6A, hU, s^U, m2A and mxG (173,290,291). Several of the r e s u l t i n g undermodified tRNA including those which accept leucine, l y s i n e , isoleucine and arginine elute at higher salt.concentrations than the f u l l y modified forms while others such as those accepting tyrosine 85. and phenylalanine elute at lower s a l t concentrations (173). The absence of ms 2£ 6A/i 6A i n these l a t t e r tRNAs probably accounts for the s h i f t to the e a r l i e r e l u t i o n p o s i t i o n . Reaction of Q containing tRNAs with CNBr, which eliminates the p o s i t i v e charge of Q, causes a dramatic s h i f t i n e l u t i o n position to higher s a l t (B. White-personal communication). The loss of the p o s i t i v e charge, i n tRNAs lacking m7G of mJA, would r e s u l t i n a tRNA eluting at a higher s a l t concentration. Conversely tRNAs de-f i c i e n t i n uridine-5-oxyacetic acid would have one less negative Val charge and should elute e a r l i e r than the mature form. If tRNA^ Val i s an undermodified form of tRNA^ , i t s elution position i s e a s i l y explained i n terms of the absence of uridine-5-oxyacetic acid. The experimental r e s u l t s described i n t h i s thesis argue against any simple r o l e for aminoacyl-tRNA i n the s p e c i f i c con-t r o l of i n d i v i d u a l tRNA biosynthesis. The r e s u l t s confirm that tRNA populations are not s t a t i c . I t would appear that changes i n t o t a l acceptance can occur without any s i g n i f i c a n t changes i n r e l a t i v e isoacceptor d i s t r i b u t i o n . An excellent example of t h i s i s seen i n the analysis of tRNA of E. c o l i S t r D discussed l a t e r i n the thesis. This would suggest that there i s a control mech-anism capable of maintaining the r e l a t i v e isoacceptor d i s t r i b u t i o n . One might ask how such a mechanism functions when several other Ser tRNAs form part of the t r a n s c r i p t . For tRNA the r e s u l t s sug-gested that the lev e l s of p a r t i c u l a r isoacceptors could be se-l e c t i v e l y increased or decreased. There was no obvious r e l a t i o n -ship between growth condition and the changes observed. I t i s 8 6 . possible that.'tRNA 0" isoacceptors are under s p e c i f i c i n d i v i d u a l control while the tRNA V a l isoacceptors are subject to quite d i f f e r e n t control mechanisms. The data are made more complicated by the presence of novel isoacceptors whose gene o r i g i n i s not known. Mechanisms for the control of tRNA biosynthesis appear to e x i s t but attempts to understand and interpret them w i l l re-quire the accumulation of considerably more data. E f f e c t of Growth Temperature on E. c o l i tRNA The r e s u l t s obtained using amino acid depletion and amino acid analogues to obtain altered tRNA isoacceptor p r o f i l e s sug-gested that the more slowly the c e l l s grew the more altered the tRNA p r o f i l e s were l i k e l y to be. Since E. c o l i grow over a wide temperature range, often at very slow growth rates (273), i t was decided that growth at extremes of growth temperature might be a useful t o o l to obtain novel tRNA isoacceptors. E. c o l i B c e l l s were therefore grown over a range of temperatures i n the Biogen as described i n Methods and the tRNA was i s o l a t e d and analyzed. As shown i n table 4, numerous changes i n amino acid accept-ance r a t i o s were observed as a function of growth temperature. In most instances t h i s r a t i o decreased suggesting that the r e l a -t i v e threonine acceptance was i n fact increasing. The percent decreases, however, were quite variable. tRNA from c e l l s grown at 44°C showed a 64% decrease i n the tyrosine to threonine acceptance r a t i o but only a 10% decrease i n the proline to threo-nine acceptance r a t i o . tRNA from c e l l s grown at 17°C showed i n -creases i n several amino acid acceptance r a t i o s including a 13% T a b l e 4. Amino a c i d a c c e p t a n c e o f c r u d e tRNA o f E. c o l i B c e l l s grown a t v a r i o u s t e m p e r a t u r e s — G r o w t h G r o w t h R a t e T e m p e r a t u r e 1 dph 17° 0.24 1.44 1.65 0.80 1.91 1.30 26 20° 0.30 1.17 1.51 0.54 1.90 1.18 52 30° 1.0 0.87 55 37° 1.0 1.28 1.57 0.65 2.10 1.19 65 41° 1.0 1.06 60 44° 0.20 0.83 1.42 0.29 1.42 0.93 62 00 88. i n c r e a s e i n t h e v a l i n e t o t h r e o n i n e a c c e p t a n c e r a t i o . No s p e c -i f i c o v e r a l l p a t t e r n i n a m i n o a c i d a c c e p t a n c e r a t i o s c a n b e d e -d u c e d f r o m t h e d a t a p r e s e n t e d i n t a b l e 4 n o r p e r h a p s i s t h e r e r e a s o n t o e x p e c t o n e . V a l R a d i o a c t i v e l y l a b e l l e d V a l - t R N A f r o m e a c h g r o w t h temp-e r a t u r e was r u n o n RPC-5 a s shown i n F i g u r e s 19-26. C o c h r o m a t o -g r a p h y w i t h V a l - t R N A V a l (B-37°) a n d V a l - t R N A V a l (NF162-Arg) i n m o s t i n s t a n c e s a l l o w s one t o r e l a t e g r o w t h c o n d i t i o n s p o s s e s s -V a l i n g s i m i l a r V a l - t R N A i s o a c c e p t o r s . A s c a n be s e e n f r o m F i g u r e s 19 a n d 2 0 , V a l - t R N A V a l (B-44°) d i f f e r s d r a m a t i c a l l y f r o m t h e V a l - t R N A V a l p r o f i l e s d e s c r i b e d t h u s f a r i n t h a t V a l - t R N A ^ a l i s n o t t h e m a j o r v a l i n e i s o a c c e p t o r . V a l - t R N A ^ 3 1 i s p r e s e n t a s i s V a l - t R N A ^ a l . V a l - t R N A ^ g i s c e r t a i n l y a b s e n t . T h e r e a r e s e v e r a l n o n d i s t i n c t p e a k s i n t h e V a l - t R N A ^ 3 1 a n d V a l - t R N A Y a l c 2a 2b r e g i o n . Two new i s o a c c e p t o r s , V a l - t R N A ^ 3 and V a l - t R N A ^ 1 a r e V a l p r e s e n t . Val-tRNA-. i s p r e s e n t i n s u c h l a r g e amounts t h a t one c a n s u g g e s t t h a t i t i s a n u n d e r m o d i f i e d f o r m o f V a l - t R N A ^ a l . F i g . 21 shows t h e RPC-5 p r o f i l e o f [ 3 H ] V a l - t R N A V a l (B-41°). The p r o f i l e s u g g e s t s t h a t t h e v a l i n e i s o a c c e p t o r s 3, 3a and 3y a r e p r e s e n t i n c e l l s g r o w n a t 41°C. I t i s i n t e r e s t i n g t h a t t h e s e c e l l s p o s s e s s e d a d o u b l i n g t i m e t h e same a s c e l l s g r o w n a t 37°C. F i g . 22 shows t h e RPC-5 " p r o f i l e o f [ 3H] V a l - t R N A V a l (B-30°) . The v a l i n e i s o a c c e p t o r d i s t r i b u t i o n o f 30°C i s e s s e n t i a l l y t h a t o b s e r v e d a t 37°C e x c e p t f o r a s l i g h t i n c r e a s e i n t h e amount o f V a l - t R N A ^ 1 . 8 9 . Figure 1 9 . The cochromatography on RPC-5 of [ 1^C]Val-tRNA (B - 3 7 ° ) and [ 3H]Val-tRNA V a l ( B - 4 4 ° ) . In t h i s experiment 10 .4 A 26o units of crude tRNA of E. c o l i B grown at 3 7 ° and containing 3 .30 x 1 0 s cpm [ ^ C l V a l (80% counting e f f i c i e n c y ) plus 7 .36 ' A 2 6 o units of crude tRNA of E. c o l i B grown at 44°C and contain-ing 6 .48 x 1 0 5 cpm [ 3H]Val (37% counting efficiency) were run on RPC-5 as described i n F i g . 5. Approximately 76% of each of the applied labels was recovered i n the gradient. The [ 3H]valine and [ 1 "*C] valine used i n the aminoacylation reactions had s p e c i f i c a c t i v i t i e s of 1300 uCi/umole and 266 uCi/umole respectively. Figure 20. The cochromatography of [ 1 **C]Val-tRNA (B-44°) and [ 3H]Val-tRNA V a l (NF162-Arg). In t h i s experiment 4.5 A 2 6o units of crude tRNA of E. c o l i B grown at 44°C and containing 1.15 x 10 s cpm of [^CjVal (80% counting e f f i c i e n c y ) plus 8.1 A 26o units of crude tRNA of E. c o l i NF162 depleted of arginine and contain-ing 7.8 x 10 s cpm [ 3H]Val (3 7% counting e f f i c i e n c y ) were run as described i n F i g . 5. Approximately 98% of each of the applied labels was recovered i n the gradient. The s p e c i f i c a c t i v i t i e s of the [ 3H]valine and [ 1^C]valine used i n the aminoacylation reactions were 1300 uCi/umole and 266 yCi/ymole respectively. 90 e_0L x wdo 0) S-i Cn -H £..oi x wcia fo,J T T U B E N O . F i g u r e 2 1 . The RPC-5 e l u t i o n p r o f i l e o f [ 3 H ] V a l - t R N A V a l (B-41°). I n t h i s e x p e r i m e n t 10.5 A 2 6 o u n i t s o f c r u d e tRNA o f E. c o l i B grown a t 41° a n d c o n t a i n i n g 8.5 x 1 0 5 cpm [ 3H] V a l ( 3 7 % c o u n t i n g e f f i c i e n c y ) w e r e r u n on RPC-5 a s d e s c r i b e d i n F i g . 5. A p p r o x i m a t e l y 70% o f t h e a p p l i e d r a d i o a c t i v i t y was r e c o v e r e d i n t h e g r a d i e n t . The s p e c i f i c a c t i v i t y o f t h e [ 3H] v a l i n e u s e d i n t h e a m i n o a c y l a t i o n r e a c t i o n was 1300 y C i / u m o i e . [3h] V a l t R N A V o ' ( B - 3 0 ° ) 4 0 ^ -O 32 -X 30 100 120 140 160 T U B E N O . Figure 22. The RPC-5 eluti o n p r o f i l e of [ 3H]Val-tRNA V a l (B-30°). In t h i s experiment 8.1 A 2 6 o units of crude tRNA of E. c o l i B grown at 30° and containing 5.3 x 10 5 cpm [ 3H]Val (37% counting ef f i c i e n c y ) were run on RPC-5 as described i n F i g . 5. Approximately 82% of the applied r a d i o a c t i v i t y was recovered i n the gradient. The s p e c i f i c a c t i v i t y of the [ 3H]valine used i n the aminoacylation reaction was 13 00 uCi/umole. 93. S i g n i f i c a n t changes i n the p r o f i l e s are observed i n c e l l s grown at 25°C or at lower temperatures. From Figures 23 and 24, i t can be seen that Val-tRNAVa"'' (B-20°) contains the commonly observed valine isoacceptors 3 and 1. This tRNA lacks the valine isoacceptor 2a3 but contains 2aa and some 2a. New valine isoacceptors 2ba and l a are observed. The valine isoacceptors 3a and 3y are present as they are i n c e l l s grown at 44°C although i n considerably d i f f e r e n t proportions. A major new valine i s o -acceptor, 33/ i s also observed i n c e l l s grown at 20°C. Val-tRNA V a l (B-17°) i s quite s i m i l a r to Val-tRNA V a l (B-20°) as can be seen i n Figures 25 and 26. Absent from these p r o f i l e s are the valine isoacceptors 2a, 2b and 2a3. Val-tRNA^ 1 i s e s s e n t i a l l y absent. The valine isoacceptors 3, 3 3 , 3y» If l a , 2aa and 2ba are present although the proportion of each i s changed. Val-tRNA^ a l i s present i n large amounts in c e l l s grown at 17°C as i t i s i n c e l l s grown at 44°C. As the doubling time of the c e l l s grown at 17°C and 44°C are very s i m i l a r i t would appear that the amount of the valine isoacceptor 3y observed i s depen-dent upon the growth rate of the c e l l s . I t has been demonstrated by Lindahl et a l . (300) that c e r t a i n tRNAs are activated or renatured by heating to 60°C i n the presence of Mg 2 +. None of the v a l i n e isoacceptor p r o f i l e s described i n t h i s thesis were altered when the crude tRNA preparations were treated i n t h i s way p r i o r to aminoacylation and RPC-5 chromato-graphy. S i m i l a r l y no changes i n isoacceptor p r o f i l e s on RPC-5 were observed when any of the l a b e l l e d Val-tRNA^ 1 samples were prepared i n the presence of the other nineteen unlabelled amino acids. F i g u r e 2 3 . The RPC-5 e l u t i o n p r o f i l e o f [ 1 * c ] V a l - t R N A V c t ' L (B-37°) an d [ 3 H ] V a l - t R N A V a l (B-20°). I n t h i s e x p e r i m e n t 9.0 A 2 6 o u n i t s o f c r u d e tRNA o f E. c o l i B g r o w n a t 37°C and c o n t a i n i n g 2.8 x 1 0 5 cpm [ 1 £ * C ] V a l ( 8 0 % c o u n t i n g e f f i c i e n c y ) p l u s 4.8 A 2 6 o u n i t s o f c r u d e tRNA o f E. c o l i B g r o w n a t 20°C and c o n t a i n i n g 4.2 x 1 0 5 cpm [ 3 H ] V a l ( 3 7 % c o u n t i n g e f f i c i e n c y ) w e r e r u n o n RPC-5 a s d e s c r i b e d i n F i g . 5. A p p r o x i m a t e l y 8 5 % o f t h e a p p l i e d [lhC] and 82% o f t h e a p p l i e d [ 3H] was r e c o v e r e d i n t h e g r a d i e n t . The [ 3H] v a l i n e a n d [ ^ C ] v a l i n e u s e d i n t h e a m i n o a c y l a t i o n r e a c t i o n s h a d s p e c i f i c a c t i v i t i e s o f 1300 u C i / u m o l e a nd 266 u C i / u m o l e r e s p e c -t i v e l y . TT- "I F i g u r e 24. The c o c h r o m a t o g r a p h y o f [ 1 4 C ] V a l - t R N A (B-20°) and [ 3 H ] V a l - t R N A V a l ( N F 1 6 2 - A r g ) . I n t h i s e x p e r i m e n t 6.25 A 2 6 0 u n i t s o f c r u d e tRNA o f E. c o l i B g r o w n a t 2 0°C and c o n t a i n i n g 1.3 x 1 0 5 cpm o f [1'*C]Val (8 0% c o u n t i n g e f f i c i e n c y ) p l u s c r u d e tRNA f r o m a r g i n i n e d e p l e t e d E. c o l i NF162 a s p e r F i g . 20 w e r e r u n a s d e s c r i b e d i n F i g . 5. A p p r o x i m a t e l y 90% o f t h e a p p l i e d [ ^ C ] and 96% o f t h e a p p l i e d [ 3H] w e r e r e c o v e r e d i n t h e g r a d i e n t . The s p e c i f i c a c t i v i t i e s o f t h e [ 3 H ] v a l i n e a n d [ 1 4 C ] v a l i n e u s e d i n t h e a m i n o a c y l a t i o n r e a c t i o n s w e r e 13 00 y C i / y m o l e a nd 266 y C i / y m o l e r e s p e c t i v e l y . Figure 25. The RPC-5 eluti o n p r o f i l e of [ 1"c]Val-tRNA (B-37°) and [ 3H]Val-tRNA (B-17°). In t h i s experiment 9.0 A 2 6 o units of crude tRNA of E. c o l i B grown at 37°C and containing 2.8 x 10 5 cpm [ ^ C l V a l (80% counting e f f i c i e n c y ) plus 7.1 A 26o units of crude tRNA of E. c o l i B grown at 17°C and containing 2.8 x 10 5 cpm [ 3H]Val (37% counting e f f i c i e n c y ) were run on RPC-5 as des-cribed i n F i g . 5. Approximately 79% of the applied [1^C] and 78% of the applied [3H] r a d i o a c t i v i t y was recovered i n the gradient. The [ 3H]valine and [1'*C] valine used i n the aminoacylation reac-tions had s p e c i f i c a c t i v i t i e s of 1300 uCi/umole and 266 uCi/umole respectively. Figure 26. The cochromatography on RPC-5 of [ 3H]Val-tRNA (B-17°) and [ 1"C]Val-tRNA V a l (NF162-Arg). In t h i s experiment 11.4 A 26o units of crude tRNA of E. c o l i B grown at 17°C and contain-ing 5.87 x 10 5 cpm [ 3H]Val (37% counting e f f i c i e n c y ) plus crude tRNA of arginine depleted E. c o l i NF162 c e l l s containing 7.0 x 101* cpm [ l l fC]Val (80% counting e f f i c i e n c y ) were run as described i n F i g . 5. Approximately 80% of each of the applied labels was recovered i n the gradient. The s p e c i f i c a c t i v i t i e s of the [3H] valine and [ 1 I fC]valine used i n the aminoacylation reactions were 1330 uCi/umole and 266 uCi/umole respectively. -_0L x U J C' : > H, 97 VO CN CD Cn •H EM K T C O Z Di > r -1_£2_J + 'at < i - CN O Z Di U 1 O CO ,_0L x w d o in CN n cn •H fe C_0L x t u d D J j . ] ^ C-OL x ^do ^ 98. To check on the generality of novel isoacceptor formation, the tRNA of the various growth temperatures was analyzed with respect to the isoacceptor d i s t r i b u t i o n s for those tRNAs accept-ing leucine, serine and threonine. Figures 27 to 29 and F i g . 13 show the RPC-5 p r o f i l e s of l a b e l l e d Leu-tRNA L e u from E. c o l i B grown at 17°C, 20°C, 37°C and 44°C. At both high and low temperatures novel leucine isoacceptors are observed. I t was shown by cochromatography that the f i r s t and the l a s t two leucine isoacceptors of the Leu-tRNA L e u (B-17°) p r o f i l e are the same isoacceptors observed at 37°C. A l l other leucine isoacceptors observed i n E. c o l i grown at 17°C appear to be novel and charac-t e r i s t i c of the extreme growth temperature. Figures 30 and 31 show the [ 3H]Ser-tRNA S e r p r o f i l e s of E. c o l i grown at 17°C and 44°C respectively. Both high and low extremes of growth tempera-ture r e s u l t i n the formation of novel serine tRNA isoacceptors. Figs. 32 to 34 show the r e s u l t s of cochromatography on RPC-5 of t 1 l tC]Thr-,tRNA T h r (B-37°) and [ 3H] Thr-tRNA T h r of c e l l s grown at 17°C, 20°C and 44°C respectively. In contrast to the results obtained for v a l i n e , leucine and serine tRNA isoacceptors, no novel threonine tRNA isoacceptors are observed i n c e l l s grown at either 17°C or 20°C. Thus not a l l tRNA populations are equally capable of giving r i s e to novel tRNA isoacceptors i n E. c o l i grown at low temperatures. There are some quantitative changes i n the r e l a t i v e threonyl isoacceptor d i s t r i b u t i o n . In agreement with previous r e s u l t s , growth at 44°C res u l t s i n the formation of novel threonine isoacceptors and as i n the case of valine isoacceptors from that growth temperature, the major Figure 27. The RPC-5 elut i o n p r o f i l e of [1''C] Leu-tRNA L e u (B-17°). In t h i s experiment 6.6 A 2 6 o units of crude tRNA of E. c o l i B grown at 17°C and containing 1.32 x 10 s cpm [ 1 4C] Leu (80% counting efficiency) were run on RPC-5 as described i n F i g . 16. Approximately 86% of the applied r a d i o a c t i v i t y was recovered i n the gradient. The s p e c i f i c a c t i v i t y of the [1''C] leucine used i n the aminoacylation reaction was 294 uCi/umole. 60 80 100 1 2 0 1 4 0 1 6 0 180 200 T U B E N O . F i g u r e 2 8 . The R P C - 5 e l u t i o n p r o f i l e o f [ 1 " c ] L e u - t R N A L e u ( B - 2 0 ° ) . I n t h i s e x p e r i m e n t 7 . 4 A 2 6 o u n i t s o f c r u d e tRNA o f E. c o l i B g r o w n a t 2 0 ° C and c o n t a i n i n g 2 . 7 x 1 0 5 cpm [lhC] L e u ( 8 0% c o u n t i n g e f f i c i e n c y ) w e r e a p p l i e d t o a s t a n d a r d a n a -l y t i c a l c o l u m n a n d e l u t e d w i t h s t a n d a r d b u f f e r s w i t h i n a l i n e a r N a C l g r a d i e n t f r o m 0 . 5 2 5 t o 0 . 9 5 M. A p p r o x i m a t e l y 7 7 % o f t h e a p p l i e d r a d i o a c t i v i t y was r e c o v e r e d i n t h e g r a d i e n t . The s p e c i f i c a c t i v i t y o f t h e [ 1 ^ C ] l e u c i n e u s e d i n t h e a m i n o a c y l a -t i o n r e a c t i o n was 2 9 4 u C i / y m o l e . 101. i 1 1 1 1 1 1 1 :—| [3H] LeutRNA U u (B-44°) 80 100 120 140 160 180 200 TUBE NO. Figure 29. The RPC-5 p r o f i l e of [ 3H] Leu-tRNA l j e u (B-44°). In t h i s experiment 3.0 A 2 6 o units of crude tRNA from E. c o l i B grown at 44°C and containing 10.0 x 10 5 cpm [ 3H]Leu (37% counting e f f i c i e n c y ) were run as described i n F i g . 16. Approx-imately 75% of the applied r a d i o a c t i v i t y was recovered i n the gradient. The s p e c i f i c a c t i v i t y of the [ 3H]leucine used in the aminoacylation reaction was 5,000 uCi/umole. 102. TUBE NO. F i g u r e 3 0. The RPC-5 p r o f i l e o f [ 3 H ] S e r - t R N A S e r (B-17°). I n t h i s e x p e r i m e n t 8.0 A 2 6 0 u n i t s o f c r u d e tRNA o f E. c o l i B grown a t ^17° and c o n t a i n i n g 7.1 x 1 0 5 cpm ( 3 7 % c o u n t i n g e f f i c i e n c y ) was r u n a s d e s c r i b e d i n F i g . 1 1 . A p p r o x i m a t e l y 90% o f t h e a p p l i e d r a d i o a c t i v i t y was r e c o v e r e d i n t h e g r a d i e n t . The [ 3H] s e r i n e u s e d i n t h e a m i n o a c y l a t i o n r e a c t i o n h a d a s p e c i f i c a c t i v i t y o f 3370 u C i / u m o l e . 103. 40 60 80 100 120 140 160 180 200 T U B E N O . Figure 31. The RPC-5 p r o f i l e of [ 3H]Ser-tRNA S e r (B-44°). In th i s experiment 3.0 A 26o of crude tRNA of E. c o l i B grown at 44°C and containing 5.0 x 10 5 cpm [ 3H]Ser (37% counting e f f i c -iency) was run as described i n F i g . 11. Approximately 84% of the applied r a d i o a c t i v i t y was recovered i n the gradient. The [ 3H]serine used i n the aminoacylation reaction had a s p e c i f i c a c t i v i t y of 3370 uCi/umole. 104, [ 3 HjThrtRNA r ' , r (&-17> [,4c] Thr tRNA T h r(B -37') 18 E 1 4 Q. u U £ 1 0 | •t— o D tO D 1 14 10 £ a IE1 x •4— "> u D O na o 0-7^ o >s •4— _o 0-5 O E 100 120 140 TUBE NO. F i g u r e 32. The c o c h r o m a t o g r a p h y o n RPC-5 o f [ 1 1*C] T h r - t R N A T n r (B-37°) a n d [ 3H] T h r - t R N A ™ (B-17°). I n t h i s e x p e r i m e n t , 3.0 A 2 6 0 u n i t s o f c r u d e tRNA o f E. c o l i B grown a t 37°C and c o n -t a i n i n g 5.7 x 101* cpm [ 1 1 + C ] T h r (80% c o u n t i n g e f f i c i e n c y ) p l u s 7.5 A 2 6 o u n i t s o f c r u d e tRNA o f E. c o l i B g r o w n a t 17°C and c o n t a i n i n g 3.1 x 10 5 cpm [ 3 H ] T h r (37% c o u n t i n g e f f i c i e n c y ) w e r e a p p l i e d t o a s t a n d a r d a n a l y t i c a l c o l u m n a n d e l u t e d w i t h i n a l i n e a r N a C l g r a d i e n t 0.50 t o 0.70 M. I n t h e g r a d i e n t , 84% o f t h e [ 1 4 C ] a n d 75% o f t h e [ 3H] r a d i o a c t i v e l a b e l s w e r e r e c o v e r e d . The [ 3 H ] t h r e o n i n e a n d [ 1 h C ] t h r e o n i n e u s e d i n t h e a m i n o a c y l a t i o n r e a c t i o n h a d s p e c i f i c a c t i v i t i e s o f 2390 u C i / u m o l e a n d 205 u C i / u m o l e r e s p e c t i v e l y . F i g u r e 3 3 . The c o c h r o m a t o g r a p h y o n RPC-5 o f [ 1 h C ] T h r - t R N A i n r (B-37°) a n d [ 3 H ] T h r - t R N A T h r (B-20°). I n t h i s e x p e r i m e n t , tRNA f r o m E. c o l i B grown a t 37° a s d e s c r i b e d i n F i g . 32 p l u s 7.2 A 2 6 o u n i t s o f c r u d e tRNA o f E. c o l i B g r o w n a t 20°C and c o n t a i n i n g 5.8 x 1 0 5 cpm [ 3 H ] T h r ( 3 7 % c o u n t i n g e f f i c i e n c y ) w e r e r u n o n an a n a l y t i c a l c o l u m n a s d e s c r i b e d i n F i g . 3 2 . I n t h e g r a d i e n t 80% o f t h e [ 1^C] a n d 75% o f t h e [ 3H] r a d i o a c t i v e l a b e l s w e r e r e c o v e r e d . The s p e c i f i c a c t i v i t i e s o f t h e amino a c i d s u s e d w e r e t h o s e o f F i g . 32. 106. 60 80 100 TUBE NO F i g u r e 34. The cochromatography on RPC-5 of [ 1 4 C ] T h r - t R N A T h r (B-37°) and [ 3 H ] T h r - t R N A T h r (B-44°). In t h i s experiment, tRNA from E. c o l i B grown a t 37° as d e s c r i b e d i n Fig.32 p l u s 9.85 A 26o u n i t s o f crude tRNA of E. c o l i B grown a t 44°C and con-t a i n i n g 6.9 x 10 s cpm [ 3H]Thr (37% c o u n t i n g e f f i c i e n c y ) were a p p l i e d t o a standard a n a l y t i c a l column and e l u t e d w i t h i n a l i n e a r NaCl g r a d i e n t 0.525 to 0.725 M. In the g r a d i e n t 87% of the [^C] and 91% of the [ 3H] r a d i o a c t i v e l a b e l s were recovered. The s p e c i f i c a c t i v i t i e s o f the amino a c i d s used were those of F i g . 32. 107. threonine isoacceptors at 44 °C growth temperature are not the commonly observed threonine isoacceptors of the 37°C growth temperature. In order to investigate further the possible o r i g i n of the novel tRNA isoacceptors several experiments were planned involv-ing supplementation of the growth media at the extreme growth temperatures with selected metal s a l t s , amino acids etc. To speed t h i s "screening" process these experiments were ca r r i e d out i n flasks i n a water bath shaker as described i n Methods. When E. c o l i B was grown up i n t h i s manner i n the same media as used i n the Biogen and at 17°C, no novel Val-tRNAVa"'" isoacceptors were observed. F i g . 35 shows [ 3H]Val-tRNA V a l (B-12°) on RPC-5 in which E. c o l i B was grown at 12°C at a growth rate of 0.08 dph i n a f l a s k . Only minor differences between t h i s p r o f i l e and that c h a r a c t e r i s t i c of growth at 37°C i n flasks are observed. Val . Also, Val-tRNA^ i s not observed i n f l a s k grown c e l l s at 37°C. These re s u l t s suggest that the novel isoaccepting tRNAs are not formed as a simple consequence of the growth rate or the growth temperature. Other factors obviously are involved. These re s u l t s argue against simple temperature i n a c t i v a t i o n of tRNA modification enzymes. They suggest that the novel tRNA i s o -acceptors form as a r e s u l t of temperature aggravation of a physiological or n u t r i t i o n a l problem (at extremes of growth temperature). E. c o l i B was grown i n flasks at 41.5°C. F i g . 36 shows [ 3H]Val-tRNA (B-41.5°) on RPC-5. The valine isoacceptor d i s t r i b u t i o n observed i n flask grown c e l l s i s very much l i k e 108. Figure 35. The RPC-5 el u t i o n p r o f i l e of [ 3H]Val-tRNA V a x (B-12°). In t h i s experiment 4.9 A 2 6 0 units of crude tRNA of E. c o l i B grown at 12°C i n flas k s as described i n methods and containing 1.74 x 10 s cpm (37% counting efficiency) were run on RPC-5 as described i n F i g . 5. Approximately 85% of the applied radio-a c t i v i t y was recovered i n the gradient. The s p e c i f i c a c t i v i t y of the [ 3H]valine used i n the aminoacylation reaction was 1300 uCi/umole. Figure 36. The RPC-5 p r o f i l e of [ 3H]Val-tRNA v a x (B-41.5°). In t h i s experiment 4.7 A 26o units of crude tRNA of E. c o l i B grown at 41.5° i n a fl a s k and containing 4.3 x 101* cpm [ 3H]Val (10% counting e f f i c i e n c y ) were run as described i n F i g . 5. Approx-imately 87% of the applied r a d i o a c t i v i t y was recovered i n the gradient. The [ 3H]valine used i n the aminoacylation reaction had a s p e c i f i c a c t i v i t y of 1300 uCi/umole. Radioactivity [3H] cpm X 10 •60T 110. that observed at the s i m i l a r temperature i n Biogen grown c e l l s (see F i g . 21). Although novel isoacceptors are formed at both high and low growth temperatures, i n fact the same novel i s o -acceptors can be formed e.g. Val-tRNA^ 3 1, the mechanisms for the formation of these novel isoacceptors are d i f f e r e n t , the low temperature results not being obtained i n f l a s k grown cultures. E. c o l i B was grown i n flasks at 42°C on the enriched medium-YT broth. F i g . 37 shows the [ 3H]Val-tRNA V a l (B-42°) p r o f i l e on RPC-5. Although the growth rate was slower than for the E. c o l i B previously grown i n f l a s k s at 4-1.5°C (Fig. 36) a considerable reduction i n the early eluting novel valine isoacceptors i s ob-served. The formation of novel isoacceptors i s therefore part-i a l l y r e v e r s i b l e at high temperature by supplementing the media with one or more factors. I t has been reported i n the l i t e r a t u r e (271) that high growth temperature (44.5°) r e s u l t s i n a methionine deficiency. Simple methionine starvation (Fig. 9) did not give the early e l u t i n g novel valine isoacceptors. This suggests that the early eluting novel v a l i n e isoacceptors are not just simple undermethylated tRNAs (although they could be that as well as being undermodified i n some other aspect). I t also suggests that methionine i s not the factor necessary to i n h i b i t formation of the early eluting novel isoacceptors. Some of the data for these l a s t experiments i s summarized i n Table 5. Table 5 shows that the valine to threonine acceptance r a t i o s for tRNA from c e l l s grown i n the range 41° to 42°C are a l l decreased r e l a t i v e to the control and these r a t i o s are i n f a c t very s i m i l a r under the s l i g h t l y d i f f e r e n t growth conditions used. The 111. Figure 37. The RPC-5 eluti o n p r o f i l e of [ 3H]Val-tRNA V a x (B-42°). In t h i s experiment 5.8 A26O units of crude tRNA of E. c o l i B grown at 42°C i n flasks on YT broth as described i n methods and containing 3.8 x 10 5 cpm [ 3H]Val (37% counting ef f i c i e n c y ) were run on RPC-5 as described i n F i g . 5. Approximately 86% of the applied r a d i o a c t i v i t y was recovered i n the gradient. The s p e c i f i c a c t i v i t y of the [ 3H]valine used i n the aminoacylation reaction was 1300 uCi/umole. Figure 38. The RPC-5 el u t i o n p r o f i l e of [ 3H]Val-tRNA V a l (W-21°). In t h i s experiment 10.0 A 2 6 0 units of crude tRNA of E. c o l i W grown at 21° and containing 6.9 x 10 5 cpm [ 3H]Val (37% counting eff i c i e n c y ) were run on RPC-5 as described i n F i g . 5. Approx-imately 88% of the applied r a d i o a c t i v i t y was recovered i n the gradient. The s p e c i f i c a c t i v i t y of the [ 3H]valine used i n the aminoacylation reaction was 13 00 uCi/umole. 113. Table 5. Amino acid acceptance of crude tRNA of E. c o l i B c e l l s grown at high temperature Growth Condition Growth Rate dph Acceptance [ ^ C l V a l Acceptance [ 1 !*C]Thr Picomoles [1!*C] Thr acceptance per A 2 6 0 unit 41°C Biogen 41.5°C Flask 42.0°C Flask + YT Broth 37°C Biogen 1.0 0.43 0.30 1.0 1.06 0.97 1.12 1.28 60 41 60 65 114. s i m i l a r i t y i n these r a t i o s may suggest that there i s a s p e c i f i c tRNA population c h a r a c t e r i s t i c of growth i n the range 41° to 42°C. To determine whether the d i f f e r e n t isoaccepting tRNAs are c h a r a c t e r i s t i c of given strains of E. c o l i , various strains were tested. E . c o l i B was obtained from an e n t i r e l y d i f f e r e n t source, grown at 20°C and shown to contain the novel v a l i n e isoacceptors. E. c o l i W was grown i n the Biogen at 21°C (0.27 dph) and the tRNA i s o l a t e d and analyzed. The valine to threonine acceptance r a t i o was shown to be 1.23 where the crude tRNA accepted 53 picomoles of [ 1 ^ C] threonine per A 2 6 o unit. This [3H] Val-tRNA^ 1 (W-21°) was run on RPC-5 as shown i n F i g . 38. The p r o f i l e ob-tained i s very s i m i l a r to that of E. c o l i B grown at 37°C i n a f l a s k . None of the early eluting novel valine isoacceptors are observed. Only the l a t e - e l u t i n g valine isoacceptor i s unusual. Work must be done to determine i f that isoacceptor i s charac-t e r i s t i c of the s t r a i n or the growth condition. I t i s i n t e r e s t -ing that a smaller but s i m i l a r peak i s observed for E . c o l i B grown i n a f l a s k at 12°C (see F i g . 35). At a growth temperature of 21°C E . c o l i B grown i n the Biogen has many novel valine isoacceptors. I t w i l l be in t e r e s t i n g to determine i f E. c o l i W forms such novel isoacceptors under more adverse growth con-d i t i o n s . Additional experimentation i s necessary to define the mech-anism (s) of formation of novel isoacceptor tRNAs at extremes of growth temperature i n the Biogen. 115. A s was i n d i c a t e d i n M e t h o d s i t was n o t m e c h a n i c a l l y f e a s i b l e t o s t e r i l i z e t h e B i o g e n . A l l o t h e r p r e c a u t i o n s w e r e t a k e n t o p r e v e n t c o n t a m i n a t i o n b y o t h e r o r g a n i s m s and c o n t a m i n a t i o n was s t r i c t l y m o n i t o r e d . I n one g r o w t h e x p e r i m e n t i t was n e c e s s a r y t o d i s c a r d t h e c e l l s b e c a u s e o f t h i s p r o b l e m . The n o v e l tRNA i s o a c c e p t o r s o b s e r v e d do n o t r e p r e s e n t tRNA f o r m s o f o t h e r o r g a n i s m s . R e l a t i n g t o t h i s p r o b l e m a number o f p o i n t s a r e s t r e s s e d : (1) The i n o c u l u m was f r e e o f c o n t a m i n a t i n g o r g a n i s m s a n d was o b t a i n e d b y g r o w i n g up c o l o n i e s f r o m s i n g l e c e l l s . (2) G r o w t h c u r v e s w e r e m o n i t o r e d . P o t e n t i a l l y c o m p o s i t e c u r v e s w e r e n o t o b s e r v e d . (3) A t t h e t i m e o f h a r v e s t i n g t h e c e l l s w e r e d i l u t e d i n s t e r i l e m e d i a a n d p l a t e d . The p l a t e s w e r e i n c u b a t e d f o r 48 h r s a n d t h e n c h e c k e d f o r t h e number o f c o l o n i e s , c o l o n y m o r p h o l o g y and s e n s i t i v i t y t o phage T7. (4) A u x o t r o p h i c s t r a i n s w e r e c h e c k e d t o d e m o n s t r a t e t h e a b s e n c e o f g r o w t h i n u n s u p p l e m e n t e d m e d i a . (5) O n l y some tRNAs g i v e r i s e t o n o v e l i s o a c c e p t o r s u n d e r e a c h a d v e r s e g r o w t h c o n d i t i o n . (6) B o t h h i g h a nd l o w g r o w t h t e m p e r a t u r e s g i v e t h e same n o v e l v a l i n e i s o a c c e p t o r s . (7) The same n o v e l v a l i n e i s o a c c e p t o r s a r e o b s e r v e d a t h i g h t e m p e r a t u r e i n s t e r i l i z e d f l a s k s a n d i n t h e n o n s t e r i l i z e d B i o g e n . A l l t h e s e p o i n t s s t r o n g l y a r g u e a g a i n s t c o n t a m i n a t i o n b e i n g a f a c t o r i n t h e s e e x p e r i m e n t s . 116. I t i s l i k e l y t h a t extremes o f growth temperature i n t e r f e r e w i t h some enzyme(s) i n v o l v e d i n the proper n u t r i t i o n a l maintenance of the c e l l . Permease enzymes i n v o l v e d i n the uptake of ions e t c . from the medium are c o n s i d e r e d l i k e l y c a n d i d a t e s . I t i s tempting to s p e c u l a t e t h a t the extremes of growth temperature a c t by a l t e r i n g l i p i d s t r u c t u r e i n the membrane thereby i n a c -t i v a t i n g c e r t a i n p r o t e i n s which are very dependent on adjacent l i p i d s f o r a c t i v i t y . I f these p r o t e i n s were l i m i t i n g under p a r t i c u l a r growth c o n d i t i o n s , then the a d d i t i o n a l a g g r a v a t i o n of growth a t extremes of temperature would r e s u l t i n c e l l s s e r i o u s l y d e f i c i e n t i n the product of the f u n c t i o n of these par-t i c u l a r p r o t e i n s . Such a d e f i c i e n c y o f , e.g. p a r t i c u l a r i o n s , would have wide ranging e f f e c t s on a number of c e l l u l a r pro-cesses i n c l u d i n g m o d i f i e d n u c l e o s i d e b i o s y n t h e s i s . E. c o l i grown i n f l a s k s a t low temperature grew more q u i c k l y than E. c o l i grown i n the Biogen a t the same low temperature suggest-i n g t h a t one o r more f a c t o r s were.more l i m i t i n g i n the Biogen than i n the f l a s k . Growth i n the Biogen and i n f l a s k s d i f f e r e d a t h i g h temperature a l s o i n t h a t growth i n the l a t t e r was much slower at any p a r t i c u l a r h i g h growth temperature. Concei v a b l y c e r t a i n key b i o s y n t h e t i c enzymes c o u l d a l s o be very temperature dependent f o r a c t i v i t y . For example, i t i s known t h a t homo-s e r i n e t r a n s - s u c c i n y l a s e the f i r s t enzyme o f the methionine b i o s y n t h e t i c pathway (271) i s s e n s i t i v e to i n a c t i v a t i o n a t 44° and the r e s u l t a n t l o s s of methionine c o u l d l i m i t the amount of methylated bases formed. I t h a s b e e n shown t h a t E. c o l i g r o w n i n a medium w i t h l e s s t h a n 10 M i r o n c o n t a i n s n o v e l tRNA i s o a c c e p t o r s (170) a s m e n t i o n e d e a r l i e r . The same n o v e l i s o a c c e p t o r s a r e o b s e r v e d i n b a c t e r i a g r o w i n g u n d e r l i m i t i n g a e r a t i o n w h e r e t h e i r o n i s p r e s e n t a s t h e i n s o l u b l e h y d r o x i d e (17 0) . S i n c e we o b s e r v e n o v e l i s o -a c c e p t o r s f o r m o s t tRNAs i n c l u d i n g t h o s e l a c k i n g m s 2 i 6 A we may b e o b s e r v i n g a s i m i l a r b u t n o t i d e n t i c a l phenomenon. I t h a s b e e n r e c e n t l y d e m o n s t r a t e d (171) t h a t g r o w t h o f E. c o l i i n l o w p h o s p h a t e m e d i a a t s l o w g r o w t h r a t e s r e s u l t s i n n o v e l Phe tRNA f o r m a t i o n . The p r e s e n c e o f t h e n o v e l i s o a c c e p t o r s i s d e p e n d e n t o n b o t h t h e p h o s p h a t e c o n c e n t r a t i o n , a n d t h e g r o w t h r a t e . The n o v e l i s o a c c e p t o r s we o b s e r v e a p p e a r t o be d e p e n d e n t o n two o r more f a c t o r s a l s o ; g r o w t h t e m p e r a t u r e and one o r more n u t r i t i o n a l f a c t o r s . C h a r a c t e r i z a t i o n o f tRNA^a"'" E x p e r i m e n t s p r e v i o u s l y d e s c r i b e d s u g g e s t e d t h a t i t was V a l n e c e s s a r y t o e s t a b l i s h t h e i d e n t i t y o f tRNA^ so t h a t c h a n g e s < i n v a l i n e i s o a c c e p t o r d i s t r i b u t i o n s o n RPC-5 c o u l d b e i n t e r -p r e t e d . The f i r s t e x p e r i m e n t s t o w a r d s t h i s end i n v o l v e d t h e c h a r a c t e r i z a t i o n o f t h e 3'OH t e r m i n a l [ 3 H ] v a l i n e o l i g o n u c l e o -t i d e o b t a i n e d f r o m RNase d i g e s t i o n o f [ 3 H ] V a l - t R N A ^ 3 1 . [ 3 H ] V a l - t R N A V a l (B) a n d [ 3 H ] V a l - t R N A V a l ( 1 6 2 - A r g ) w e r e p r e p a r e d i n t h e s t a n d a r d manner and r u n i n d i v i d u a l l y on RPC-5 a s d e s -c r i b e d i n M e t h o d s . A l i q u o t s w e r e c o u n t e d on a s c i n t i l l a t i o n c o u n t e r and t h e r a d i o a c t i v i t y d i s t r i b u t i o n o n t h e c o l u m n s d e t e r -m i n e d . B a s e d u p on t h e s e r e s u l t s t h e i n d i v i d u a l Val-tRNA^ 3"'", 118. V a l - t R N A ^ 0 " 1 " a n d V a l - t R N A ^ " 1 " i s o a c c e p t o r s w e r e p o o l e d and p r e c i p i -t a t e d . The i n d i v i d u a l i s o a c c e p t o r s w e r e t h e n r e s u s p e n d e d i n b u f f e r and t r e a t e d w i t h RNase T^ a s d e s c r i b e d i n M e t h o d s . The o l i g o n u c l e o t i d e s w e r e c h r o m a t o g r a p h e d on D E A E - c e l l u l o s e and e l u -t e d w i t h a n ammonium f o r m a t e g r a d i e n t a s d e s c r i b e d i n M e t h o d s . The s e q u e n c e s o f t h e common v a l i n e i s o a c c e p t o r s (208) a r e s u c h t h a t t h e v a l i n e i s o a c c e p t o r s 2a a n d 2b b o t h g i v e r i s e t o a 3'OH t e r m i n a l p e n t a n u c l e o t i d e f r a g m e n t (CpApCpCpA) w h i l e t h e m a j o r v a l i n e i s o a c c e p t o r 1 y i e l d s a 13 n u c l e o t i d e 3'OH t e r m i n a l f r a g m e n t (UpCpApUpCpApCpCpCpApCpCpA) upon t r e a t m e n t w i t h RNase T^. T h e s e a r e e a s i l y s e p a r a t e d o n D E A E - c e l l u l o s e a s shown i n F i g . 3 9. The p e n t a n u c l e o t i d e f r a g m e n t e l u t e s a t a p p r o x i m a t e l y 0.09 M ammonium f o r m a t e pH 4.5 i . e . a b o u t t u b e 70 i n F i g . 39 a n d t h e 13 n u c l e o t i d e f r a g m e n t e l u t e s a t a b o u t 0.20 M ammonium f o r m a t e pH 4.5 i . e . a b o u t t u b e 150 i n F i g . 39. The 3*OH t e r m i n a l V a l f r a g m e n t o f tRNA^ a p p e a r s t o be t h e same s i z e a s t h e f r a g m e n t V a l f r o m tRNA^ . A t pH 4.5, RNase T^ c l e a v e s s l o w l y a f t e r A s , e s p e c i a l l y t h o s e t h a t a r e i n s i n g l e s t r a n d e d r e g i o n s o f RNA a n d h e n c e e a s i l y a c c e s s i b l e (R.C. W a r r i n g t o n - u n p u b l i s h e d r e s u l t s ) . The f i r s t m a j o r p e a k o f f t h e c o l u m n s o f F i g . 39 i s p r o b a b l y C p C p A - [ 3 H ] V a l . E v i d e n c e f r o m c h r o m a t o g r a p h y o f t h i s e a r l y e l u t i n g m a t e r i a l o n D E A E - c e l l u l o s e u r e a c o l u m n s s u g g e s t s t h a t t h i s f r a g m e n t c a n n o t be a n y l a r g e r t h a n a t r i n u c l e o t i d e ( b a s e d u p o n i t s c a l c u l a t e d c h a r g e ) . A t h i g h e r RNase T^ l e v e l s , a d d i t -V a 1 i o n a l c l e a v a g e s a f t e r A s a r e o b s e r v e d f o r [ 3 H ] V a l - t R N A ^ g i v i n g r i s e t o two a d d i t i o n a l [ 3 H ] V a l - o l i g o n u c l e o t i d e f r a g m e n t s o f p r o b a b l e s e q u e n c e UpCpApCpCpCpApCpCpA a n d CpCpCpApCpCpA. Figure 39. DEAE-cellulose chromatography of RNase digests of Veil i n d i v i d u a l [ 3H]Val-tRNA isoacceptors. Individual isoacceptors were prepared as described i n Methods and incubated with RNase as indicated. Top: 8.0 x 10" cpm [ 3H]Val-tRNA^ a l (10% counting efficiency) i n a t o t a l volume of 1.8 ml of digestion buffer to which was added 100 Sankyo units of RNase T^. The mixture was incubated for 4 hrs at 37°C and run on the column as described i n Methods. Middle: 6.5 x 10 "* cpm [ 3H] Val-tRNA* (10% counting e f f i c i e n c y ) i n a t o t a l volume of 1.5 ml of digestion buffer to which was added 50 Sankyo units of RNase T^. The mixture was incubated for 4 hrs at 37°C and run on the column as described i n Methods. Bottom: 14.8 x 10k cpm [ 3H]Val-tRNA*^ (10% counting e f f i c i e n c y ) i n a t o t a l volume of 1.5 ml of digestion buffer to which was added 50 Sankyo units of RNase T^. The mixture was incubated for 4 hrs at 37° and run on the column as described i n Methods. Figure 39 120. Hj Va!-RNase T] Oligonucleotides on D E A E cellulose 121. In an analogous experiment, i t has been shown that the same two Val addi t i o n a l fragments can be obtained from [3H] Val-tRNA^ This strongly suggests that not only are the 3' terminal o l i g o -nucleotides of RNase treated valine isoacceptors 1 and 3 i d e n t i -c a l i n si z e , they probably also have an i d e n t i c a l sequence. This i n turn argues that they are both transcribed from the Val same gene(s) and that Val-tRNA^ i s probably an undermodified form of Val-tRNA* . V a l P u r i f i c a t i o n of tRNA* To te s t t h i s p o s s i b i l i t y further, the nucleoside analysis Val Val of tRNA^ was required. Relatively pure tRNA^ was iso l a t e d as described i n Methods. The RPC-5 el u t i o n p r o f i l e for absorb-ance at 260 nm and [ 1 "*C] valine acceptance are shown i n F i g . 40. for the f i n a l step of the p u r i f i c a t i o n procedures. The column was assayed by aminoacylating 25 ul aliquots from each tube i n a standard charging experiment having a t o t a l reaction volume of 100 y l . Aliquots of 50 y l were pipetted onto f i l t e r paper discs a f t e r 20 min incubation and the acid i n s o l -uble [ 1 I fC] valine determined. The charging conditions used i n -sured complete saturation of the tRNA with the la b e l l e d amino acid. The s p e c i f i c a c t i v i t y of the [ l l tC]valine used i n the amino-acylation reaction was 209 yCi/ymole. The e f f i c i e n c y of radio-active counting of the [^C]valine on f i l t e r paper discs was found to be approximately 60% at the channel settings used. The s p e c i f i c a c t i v i t y of the peak f r a c t i o n #95 was 1733 picomoles of [1'tC] valine acceptance per A 26o unit. Based upon 1 2 2 . 0-18 0-16 0-14 £ c O 0-12 O CN <D 0-10 u c D -Q v_ O i/> < 0-08 0-06 0-04 0-02 h-RPC-5 Chromatography of tRNA Val 90 110 130 150 TUBE NO. CM I O £ a u 7r 3 •+-o g D U 0-7 ^ 0-6 ° Val Figure 40. F i n a l step i n the p u r i f i c a t i o n of tRNA^ . In t h i s experiment 47.8 A 26o units of tRNA enriched for tRNA^ 3 1 wererrunon a 2.4 x 45 cm RPC-5 column (50 mesh sieve size) and eluted with 2 1. of buffer containing 10 mM t r i s - H C l pH 7.0, 1 mM 2-mercaptoethanol within a l i n e a r NaCl gradient of 0.50 to 0.65. The f r a c t i o n size was 10 ml and the column flow rate was 180 ml/hr. The s o l i d l i n e indicates the absorbance p r o f i l e at 260 nm while the dotted l i n e refers to the [1HC]valine acceptance i n a standard aminoacylation reaction. the d i s t r i b u t i o n of amino acid acceptance i n r e l a t i o n to the absorbance at 260 nm, tubes 91 to 100 inc l u s i v e were pooled and Val considered a pure sample of tRNA^ . The t o t a l y i e l d was 11.4 A 2 6 0 units a f t e r p r e c i p i t a t i o n with ethanol and c o l l e c t i o n of the p r e c i p i t a t e by M i l l i p o r e f i l t r a t i o n . Val Spectral C h a r a c t e r i s t i c s of tRNA^ Val The tRNA3 was dissolved i n 1.0 ml of d i s t i l l e d H 20 and an absorbance spectrum determined as shown i n F i g . 41. The spectrum was compared with spectra of commercial crude E. c o l i tRNA and crude tRNA of control E. c o l i B growth experiments. S i g n i f i c a n t spectral differences were observed i n the 330 to 340 nm region. Absorbance i n t h i s region i s due to the presence of the minor nucleoside s^U. The absence of any s p e c i f i c absorb-Val ance peak i n t h i s region i n p u r i f i e d tRNA^ strongly suggests that s^U i s not present i n tRNA^ a x. A s i g n i f i c a n t absorption i s seen i n t h i s region for both commercial tRNA and E. c o l i B crude tRNA. The A 3 3s to A 2 6 o r a t i o s for these l a t t e r two samples are s i m i l a r to those reported i n the l i t e r a t u r e (173,301). Val Q u a l i tative Nucleotide Analysis of tRNA^ In a subsequent experiment 3.0 A 2 6 0 units of the p u r i f i e d Val tRNA3 were RNase treated and a preliminary nucleotide analysis c a r r i e d out as described i n Methods. As shown i n F i g . 42, pres-ent are the nucleotides Tp, Ap, Cp, Gp, Up, m7Gp, ¥p, a breakdown product of m7Gp (B.N. White-personal communication) and the nucleoside A of the 3' terminal CpCpA sequence. Present i n any 124. Figure 41. U l t r a v i o l e t absorption spectra of E. c o l i tRNA i n 1 ml d i s t i l l e d H 20 (1 cm path length) Commercial tRNA; 12.0 A 2 6 o units/ml Crude tRNA from E. c o l i B control; 12.0 A 2 6 o units/ml V a l P u r i f i e d tRNAI. ; 11.4 A 2 6 0 units/ml F i g u r e 41 310 326 330 340 350 360 wavelength (nnn) 2 n d dimension @ fluorescent in acid O not fluorescent in acid F i g u r e 42. Two dimensional t h i n l a y e r chromatogram of n u c l e o t i d e V a l d i g e s t of tRNA^ . 3.0 A 2 6 o of d i g e s t e d m a t e r i a l was a p p l i e d to 10 cm x 10 cm c e l l u l o s e on g l a s s p l a t e s and chromatographed as d e s c r i b e d i n Methods. 127. s i g n i f i c a n t amounts are the nucleotides s^Up and m6Ap. I t i s not possible to comment on the presence or absence of Vp (the nucleo-tide of uridine-5-oxyacetic acid) or hUp because of the d i s t o r t -ion of the Up spot p a r t i c u l a r l y i n the second dimension of chromatography. The absence of s^Up confirms the e a r l i e r re-sults of spectral analysis. Quantitative nucleoside analysis was subsequently performed by the Randerath t r i t i u m l a b e l l i n g technique. Val Randerath Nucleoside Analysis of tRNA^ Val The t o t a l nucleoside analysis of tRNA^ was carr i e d out by Randerath t r i t i u m l a b e l l i n g techniquesas described i n Methods. For comparison purposes, the nucleoside composition of a g i f t . . Val of p u r i f i e d tRNA^ was determined i n a p a r a l l e l experiment. Fi g . 43 shows the res u l t s of the fluorograms of these experiments. Several q u a l i t a t i v e differences are noted. tRNA^^ lacks the nucleosides m6A and V (uridine-5-oxyacetic acid) present i n Val tRNA^aJ" and X (1-[3-amino-3-carboxypropyl] uridine),. present i n tRNA^ 1 and tRNA^"*". The reacted nucleoside spot for V was eluted and shown to have one negative charge by paper e l e c t r o -phoresis at pH 7.0. Differences i n the uridine spots are due to the use of d i f f e r e n t batches of thi n layer chromatography plates. Such differences have been noted previously i n the l i t e r a t u r e (304). Individual spots were cut out and the radio-active nucleoside t r i o l s eluted and counted as described by Randerath. A quantitative nucleoside analysis was obtained for tRNA^3"*" and tRNA^3^ as shown i n Table 6. The experimental Figure 43. Randerath nucleoside analysis of tRNA^ a l (A) and tRNA^ a x (B). Randerath nucleoside analysis was carried out as described i n methods. Each TLC plate was spotted with 10 y l of the f i n a l reaction mix (.06 to .10 A 2 6 o units) and eluted f i r s t i n solvent F and subsequently i n solvent G (260). The fluorograms were developed af t e r 72 hours exposure. Table 6. Nucleoside Analysis of tRNA 1 2a 2b 3 3 1 Nucleoside predicted predicted predicted predicted observed observed A 14 13 16 15 13.8 14.0 C 23 21 18 23 23.0 23.1 G 23 25 22 23 22.8 22.1 U + s4U 10 10 13 11 11.5 10.4 T 1 . 1 1 1 1.0 0.8 1 1 1 1 0.9 0.9 hU 1 4 4 1 2.8 1.0 m7G 1 1 1 1 0.6 0.6 m6A 1 - - - 0.8 V 1 - - - - 0.9 X • - 1 1 - -V - uridine-5-oxy-acetic acid X - 1-(3-amino-3-carboxypropyl) uridine predicted: means the # of nucleoside residues per tRNA molecule based upon the known or postulated sequence. observed: means the # of nucleosides experimentally determined based upon the average of three measurements and calculated for a tRNA molecule of 76 nucleoside residues. 130. values reported are the average of three determinations. The m7G spot has not been corrected for degradation to g l y c e r o l . The chemistry of the Randerath technique i s such that s i s degraded to U during the course of the reaction. I t has been shown previously, however, that there i s l i t t l e or no s i n Val tRNA^ . The experimentally determined nucleoside analysis of Val . tRNA^ i s very close to that predicted based upon the known sequence. The only s i g n i f i c a n t difference i s that there i s approx-imately one less G residue per tRNA than predicted. The accuracy Val of the determination of the nucleoside content of tRNA-^  i n -creases confidence i n the experimental values obtained for Val tRNA3 . A column i n Table 6 has been c a l l e d 3 predicted. This column refers to the anticipated nucleoside composition i f tRNA^3"*" i s an undermodif ied form of tRNA^ a"*" lacking the modified nucleoside sk\J, m6A and V (uridine 5-oxyacetic acid) and having instead U, A and U respectively at those positions. As can be seen from the table, the data for C, G, U, T, m7G, m6A and \\) of tRNA^3''' experimental i s consistent with tRNA^ 3"*" predicted as Val previously defined except that tRNA^ has approximately one too few A residues and nearly two extra hU residues (2.9, 2.7, 2.8 Val average of 2.8). The hU data would argue that tRNA^ i s a Val Val precursor of either tRNA0 or tRNA-, . However, the data for A3. AD tRNA^"*" for A residues i s very d i f f e r e n t from that for tRNA^"*" predicted while the data for G residues i s very d i f f e r e n t from that for tRNA^ a l predicted. The nucleoside analysis of tRNAY a l £ a J Val experimental seems most si m i l a r to that for tRNA^ predicted. 131. T h e R N a s e T ^ 3 ' O H t e r m i n a l o l i g o n u c l e o t i d e a n a l y s i s i n d i c a -V a l t e d t h a t t R N A ^ h a d a t e r m i n a l o l i g o n u c l e o t i d e o f t h e s a m e s i z e V a l a n d s e q u e n c e a s t R N A ^ . S u b s e q u e n t n u c l e o s i d e a n d n u c l e o t i d e a n a l y s i s s h o w e d t h a t t R N A ^ a x d i f f e r s i n t o t a l n u c l e o s i d e c o m -. . V a l p o s i t i o n f r o m t R N A ^ b y t h e a b s e n c e o f t h e m o d i f i e d n u c l e o s i d e s s ^ U , V a n d m 6 A . I t a p p e a r s t h a t t R N A ^ a x c o u l d b e a n u n d e r -V a l m o d i f i e d f o r m o f t R N A ^ w h i c h a c c u m u l a t e s u n d e r a d v e r s e g r o w t h c o n d i t i o n s . H o w e v e r , a p o s s i b i l i t y w h i c h i s n o t e l i m i n a t e d b y t h e p r e s e n t V a l . d a t a i s t h a t t R N A ^ i s t r a n s c r i b e d f r o m a u n i q u e g e n e w h i c h i s v e r y s i m i l a r t o t h a t f o r t R N A ^ a x a n d w h i c h i s d e r e p r e s s e d d u r i n g a d v e r s e g r o w t h c o n d i t i o n s . T h i s p o s s i b i l i t y s h o u l d b e d i s -p r o v e d b y f u r t h e r e x p e r i m e n t a t i o n . t R N A o f E . c o l i B s t r D I t h a s b e e n r e p o r t e d i n t h e l i t e r a t u r e (320) t h a t s t r e p t o -m y c i n d e p e n d e n t E . c o l i h a v e a n a l t e r e d a m i n o a c i d m e t a b o l i s m s u c h t h a t t h e y e x c r e t e u p t o 10% o f t h e i r t o t a l c a r b o n s o u r c e a s v a l i n e . A n y t h e o r y w h i c h i n c l u d e s s o m e f o r m o f a m i n o a c i d c o n t r o l o f t R N A b i o s y n t h e s i s w o u l d p r e d i c t c h a n g e s i n t h e t R N A p o p u l a t i o n o f s u c h a n o r g a n i s m . I f v a l i n e i s p a r t ( e . g . V a l -t R N A ^ 1 ) o f a f e e d b a c k r e p r e s s o r c o m p l e x f o r t R N A V a x t h e n a d e c r e a s e i n t h e [ l l f C ] v a l i n e t o [ 1 ^C] t h r e o n i n e a c c e p t a n c e r a t i o i s p r e d i c t e d f o r s t r e p t o m y c i n d e p e n d e n t E . c o l i . T h e s t r e p t o -m y c i n d e p e n d e n t E . c o l i B s t r a i n w a s g r o w n u p i n o n e l i t e r f l a s k s a s d e s c r i b e d i n m e t h o d s a n d t h e t R N A a n a l y z e d . A s s h o w n i n T a b l e 7 v e r y d r a m a t i c d i f f e r e n c e s i n t h e a m i n o a c i d a c c e p t a n c e Table 7. Amino acid acceptance of crude tRNA of E. c o l i B str 1 133. r a t i o s r e l a t i v e to the control parent s t r a i n were observed. Of a l l the acceptance r a t i o s measured, only that for tyrosine re-mained unaltered. A 51% decrease i n the [ l t fC]valine to [^C] threonine acceptance r a t i o was observed. In contrast, a 55% increase i n the [ 1^C]arginine to [ 1^C]threonine acceptance was noted. Since many acceptance r a t i o s changed i t i s d i f f i c u l t to r a t i o n a l i z e how any one of them was changed s p e c i f i c a l l y . It i s i n t e r e s t i n g that such a variety of differences are a l l the r e s u l t of a single point mutation. Since such large changes i n amino acid acceptance r a t i o s were observed i t was important to determine the tRNA isoacceptor d i s t r i b u t i o n s on RPC-5. Figs. 44-4 6 show the re s u l t s of such experiments. A comparison of the RPC-5 el u t i o n p r o f i l e s of [ 3H] Val-tRNA V a l, [ l l*C]Leu-tRNA L e u and [ 3H]Ser-tRNA S e r of E. c o l i B s t r D with the corres-ponding p r o f i l e s for E. c o l i B (figures 1, 16 and 11 respectively) suggests that,,while the corresponding p r o f i l e s are not i d e n t i c a l , they are remarkably s i m i l a r . The i n d i v i d u a l isoacceptor c o n t r i -bution to the t o t a l acceptance was i n each instance determined by measuring the peak area of each isoacceptor. Although a 51% decrease i n the [1'*C] valine acceptance to [ 1 ^ C] threonine accept-ance r a t i o was noted e a r l i e r , on RPC-5 there are only small differences i n the r e l a t i v e d i s t r i b u t i o n of the major valine isoacceptors 1, 2a and 2b. None of the minor valine isoacceptor 3 i s observed i n [ 3H]Val-tRNA V a l E. c o l i B s t r D tRNA. S i m i l a r l y , while a 26% increase i n the [ 1 "*C] leucine acceptance to [1'*C] threonine acceptance r a t i o i s observed, only s l i g h t differences i n the leucine isoacceptor d i s t r i b u t i o n on RPC-5 are noted. F i g u r e 44. The RPC-5 e l u t i o n p r o f i l e o f [ 3 H ] V a l - t R N A V a ± E. c o l i B s t r D . I n t h i s e x p e r i m e n t 5.9 A 26o u n i t s o f c r u d e E. c o l i B s t r D tRNA c o n t a i n i n g 5.7 x 10 **• cpm [ 3 H ] V a l ( 1 0 % c o u n t i n g e f f i c i e n c y ) w e r e a p p l i e d t o a s t a n d a r d a n a l y t i c a l c o l -umn a n d e l u t e d w i t h t h e s t a n d a r d b u f f e r s w i t h i n a l i n e a r N a C l g r a d i e n t o f 0.50 t o 0.65 M. I n t h e g r a d i e n t 2.5 x 10** cpm ( 5 % c o u n t i n g e f f i c i e n c y ) was r e c o v e r e d i . e . 88% o f t h a t a p p l i e d . The [ 3 H ] v a l i n e u s e d i n t h e a m i n o a c y l a t i o n r e a c t i o n h a d a s p e c -i f i c a c t i v i t y o f 1300 u C i / u m o l e . CO I 22 20 18 1 6 E D_ 14 > 1 0 ° ft .2 O I I I 1 [Mc] Leu t R N A t 0 U CB - S t r D ) 100 120 140 TUBE NO. -|0-6_D o E 180 200 Leu Figure 45. The RPC-5 elut i o n p r o f i l e of [1^C]Leu-tRNA E. c o l i B s t r D . In t h i s experiment 6.3 A 2 6o units of crude E. c o l i B s t r D tRNA containing 2.9 x 10 5 cpm [ 1^C]Leu.(76% counting efficiency)were applied to a standard a n a l y t i c a l column and eluted with the standard buffers within a l i n e a r NaCl gradient of 0.50 to 0.95 M. Approximately 77% of the applied r a d i o a c t i v i t y was recovered i n the gradient. The [ 1^C]leucine used i n the aminoacylation reaction had a s p e c i f i c a c t i v i t y of 294 uCi/umole. Figure 46. The RPC-5 elution p r o f i l e of [ 3H]Ser-tRNA & e r E. c o l i B s t r D . In t h i s experiment 5.6 A 26o units of crude E. c o l i B s t r D tRNA containing 3.5 x 10 5 cpm [ 3H]Ser (10% counting efficiency)were applied to a standard a n a l y t i c a l column and eluted with the standard buffers within a l i n e a r NaCl gradient of 0.45 to 1.50 M. Approximately 76% of the applied r a d i o a c t i v i t y was recovered i n the gradient. The [ H ] s e r i n e used i n the aminoacylation reaction had a s p e c i f i c a c t i v i t y of 3370 uCi/umole. 137. I n n e i t h e r i n s t a n c e c a n t h e o v e r a l l c h a n g e i n t h e a m i n o a c i d a c c e p t a n c e r a t i o be e x p l a i n e d i n t e r m s o f t h e a l t e r e d s y n t h e s i s a n d / o r d e g r a d a t i o n o f p a r t i c u l a r i s o a c c e p t o r s . I t i s p a r t i c u l a r l y i n t e r e s t i n g t h a t t h e r e l a t i v e i s o a c c e p t o r d i s t r i b u t i o n i s s o w e l l m a i n t a i n e d . T h i s i s a n i m p o r t a n t c o n c e p t t h a t w o u l d h a v e t o be e x p l a i n e d i n a n y t h e o r y p o s t u l a t i n g s p e c i f i c c o n t r o l o f tRNA b i o s y n t h e s i s . Some d i f f e r e n c e s i n t h e s e r i n e i s o a c c e p t o r d i s t r i b u t i o n s a r e o b s e r v e d . I n p a r t i c u l a r , t h e r e l a t i v e d i s -t r i b u t i o n b e t w e e n t h e two m a j o r s e r i n e i s o a c c e p t i n g p e a k s i s d i f f e r e n t a n d t h e f i n a l e l u t i n g s e r i n e i s o a c c e p t o r o f [ 3 H ] S e r -S e r D tRNA E. c o l i B s t r i s c o n s i d e r a b l y d i m i n i s h e d . T h e s e c h a n g e s h o w e v e r do n o t r e a d i l y e x p l a i n t h e 1 2 % i n c r e a s e i n t h e [ 1 I fC] s e r i n e a c c e p t a n c e t o [ 1 k C ] t h r e o n i n e a c c e p t a n c e r a t i o w h i c h was o b s e r v e d . R e c e n t e x p e r i m e n t s i n a n o t h e r l a b h a v e shown t h a t a m ino a c i d e x c r e t i o n i n s t r e p t o m y c i n d e p e n d e n t E. c o l i K12 i s v e r y s e n s i t i v e t o t h e c o n c e n t r a t i o n o f F e + + i n t h e medium (W.J. P o l g l a s e - p e r s o n a l c o m m u n i c a t i o n ) . V a l i n e i s e x c r e t e d i n F e + + c o n t a i n i n g m e d i a ( 0 . 1 m i c r o m o l a r ) a n d g l u t a m a t e i s e x c r e t e d i n F e + + d e f i c i e n t m e d i a . I t h a s n o t b e e n shown i f s u c h r e s u l t s a p p l y t o E. c o l i B. As t h e F e + + o f t h e g r o w t h m e d i a u s e d i n my e x p e r i m e n t s i s n o t p r e c i s e l y known, i n t e r p r e t a t i o n o f t h e r e s u l t s may be d i f f i c u l t . F u r t h e r w o r k i s n e c e s s a r y t o c h e c k o n a n y d e p e n d e n c e o f t h e tRNA p o p u l a t i o n i n s t r e p t o m y c i n d e p e n d e n t c e l l s o n t h e F e + + c o n c e n t r a t i o n o f t h e medium. I t i s a l s o n e c e s s a r y t o o b t a i n i n f o r m a t i o n o n tRNA G"'" u u n d e r t h e v a r i o u s g r o w t h c o n d i t i o n s . E x p e r i m e n t s s h o u l d be 138. done to check for any changes i n the tRNA population as a func-t i o n of the streptomycin l e v e l s of the media. Gel Electrophoresis of Crude E. c o l i tRNA Although the E. c o l i growth and subsequent tRNA extraction procedures used as described i n Methods are not optimal for the i s o l a t i o n of precursor tRNAs having extra nucleotide sequences, the crude tRNAs i s o l a t e d were analyzed by polyacrylamide gel electrophoresis to check for such precursors. In these studies electrophoresis i n 15% polyacrylamide gels i n 6 M urea gives good resolution of 4-6S RNAs. The major differences seen i n such gels (Figures 47 to 49) between crude tRNA preparations from c e l l s grown at the various temperatures under several growth conditions are seen i n the 4.5S-5S RNA regions. In the 4S, or tRNA region, the background of unchanged tRNAs i s so great that the changes i n p a r t i c u l a r species observed by RPC-5 chromatography are not detectable. Growth of E. c o l i B at low temperatures re s u l t s i n the accumulation of material i n the 5S region. This i s noticeable i n F i g . 47 for the 17°C and 20°C s l o t s , s l o t s 2 to 5 of F i g . 48 and s l o t 4 of F i g . 49. The i d e n t i t y of t h i s RNA has not yet been established. A s i g n i f i c a n t amount of the minor nucleoside i s present i n the RNA of t h i s region (I.C. Gillam-personal communication), thus suggesting that some of the material i s tRNA precursor. The RNA of t h i s region i s approximately the same size as the 3 group precursor tRNAs reported by Dijk and Singhal (38). 1 7 ° 2 0 ° 3 7 ° 4 4 ° Figure 47. Acrylamide g e l e l e c t r o p h o r e s i s of crude tRNA prep-a r a t i o n s from E. c o l i B grown at 17°, 20°, 37° and 44°C. Gels were run and s t a i n e d as described i n Methods. Arrows i n d i c a t e the p o s i t i o n s to which markers migrated. 140. 1 2 3 4 5 6 F i g u r e 48. A c r y l a m i d e g e l e l e c t r o p h o r e s i s o f c r u d e tRNA p r e p -a r a t i o n s o f E. c o l i . The 5S and 4.5S m a r k e r p o s i t i o n s a r e i n d i c a t e d by t h e u p p e r a n d l o w e r a r r o w s r e s p e c t i v e l y . S l o t 1. tRNA f r o m E. c o l i B grown a t 42°C i n f l a s k s on YT b r o t h . S l o t 2. tRNA f r o m E. c o l i W g r o w n a t 21°C. S l o t 3. tRNA f r o m E. c o l i B g r o w n a t 21.5°C i n YT b r o t h . S l o t 4. tRNA f r o m E. c o l i B g r o w n a t 20.3°C. S l o t 5. tRNA f r o m E. c o l i B g r o w n a t 20°C. S l o t 6. C o m m e r c i a l tRNA o f E. c o l i B. 1 4 1 . F i g u r e 49. A c r y l a m i d e g e l e l e c t r o p h o r e s i s o f c r u d e tRNA p r e p -a r a t i o n s o f E. c o l i B. The 5S a n d 4.5S m a r k e r p o s i t i o n s a r e i n d i c a t e d b y t h e u p p e r a n d l o w e r a r r o w s r e s p e c t i v e l y . S l o t 1. C o m m e r c i a l tRNA o f E. c o l i B. S l o t 2. tRNA f r o m E. c o l i B g r o w n a n a e r o b i c a l l y a t 37°C. S l o t 3. tRNA f r o m E. c o l i B g r o w n a t 41.5°C i n a f l a s k . S l o t 4. tRNA f r o m E. c o l i B g r o w n a t 21.5°C. S l o t 5. tRNA f r o m E. c o l i B g r o w n a t 44°C t o t h e s t a t i o n a r y s t a t e . S l o t 6. tRNA f r o m E. c o l i B g r o w n a t 43.5°C. 142. Growth at high temperature results i n a diminished amount of material i n the 5S region as i s seen i n F i g . 47 s l o t 44° and F i g . 4 9 sl o t s 3 and 6. Our gel electrophoresis experiments detect n e g l i g i b l e accumulation of RNAs running between 6 and 16S RNA i n E. c o l i grown at various temperatures. These re s u l t s are very preliminary. Work i s i n progress to quantitate the amount of material i n the various regions and to characterize i n d i v i d u a l gel bands. I t w i l l also be necessary to i s o l a t e crude tRNA from the various growth conditions by other common extraction pro-cedures designed to extract uncleaved precursor tRNAs i n high y i e l d . 1 4 3 . Conclusions The r e s u l t s obtained i n the experiments described i n t h i s thesis argue against any simple role for aminoacyl-tRNA i n the s p e c i f i c control of i n d i v i d u a l tRNA biosynthesis. tRNA popula-tions as measured by amino acid acceptance were shown not to be s t a t i c but rather to change as a function of growth conditions. I t was not possible to c o r r e l a t e i n any simple way the changes i n tRNA populations observed with the type of growth condition causing them. I t i s concluded that there are two or more control mechanisms involved i n regulating tRNA populations. The control mechanism observed most often brings about changes i n t o t a l amino acid acceptance while maintaining a constant r e l a t i v e isoacceptor d i s t r i b u t i o n . A second control mechanism can bring about changes i n t o t a l amino acid acceptance by a l t e r i n g the amounts of par-t i c u l a r isoacceptors thus changing the r e l a t i v e isoacceptor d i s -Ser t r i b u t i o n . tRNA l e v e l s appear to be subject to both control mechanisms. tRNA V a l and tRNA L e u are regulated by the f i r s t c o n t r o l mechanism but the presence of large amounts of novel isoacceptors of unknown gene o r i g i n does not allow us to deter-mine i f p a r t i c u l a r growth conditions r e s u l t i n the second control mechanism becoming operative f o r these tRNAs. I t may be that some tRNAs are c o n t r o l l e d by both mechanisms while others are subject to control by only one of the two mechanisms. Maintenance of r e l a t i v e isoacceptor d i s t r i b u t i o n requires that a l l the genes are c o n t r o l l e d or recognized by the same re-pressor. I t i s possible that tRNAs could be divided into groups 144. of t r a n s c r i p t i o n u n i t s each having i t s own r e p r e s s o r . I t i s very l i k e l y (P. P r i m a k o f f - p e r s o n a l communication) t h a t s e v e r a l d i f f e r -ent tRNAs are t r a n s c r i b e d t o g e t h e r i n s i n g l e u n i t s . Maintenance of r e l a t i v e i s o a c c e p t o r d i s t r i b u t i o n r e q u i r e s t h a t s e v e r a l t r a n s c r i p t i o n u n i t s having tRNAs f o r d i f f e r e n t amino a c i d s must a l l be c o - o r d i n a t e l y c o n t r o l l e d . T h i s would e x p l a i n why groups of changes i n amino a c i d acceptances c o u l d be observed,(when onl y the f i r s t c o n t r o l mechanism d e s c r i b e d i s o p e r a t i v e ) . One must a l s o ask what f a c t o r ( s ) determines r e l a t i v e i s o a c c e p t o r d i s t r i b u t i o n . Can gene number and d i f f e r e n t i a l r a t e s of degrada-t i o n e x p l a i n the r e l a t i v e amounts of i s o a c c e p t o r s observed and/or i s RNA polymerase a f f i n i t y f o r d i f f e r e n t promoters f o r each i s o a c c e p t o r coupled w i t h i n t e r a c t i o n w i t h a common r e p r e s s o r r e s p o n s i b l e f o r the r e l a t i v e l e v e l s o f the i s o a c c e p t o r s observed? The r e s u l t s o f t h i s t h e s i s g i v e s e v e r a l u s e f u l s t a r t i n g p o i n t s f o r f u r t h e r i n v e s t i g a t i o n o f c o n t r o l mechanisms f o r tRNA b i o -s y n t h e s i s . 145. Blb l i o graphy (1) Lengyel, P. and S P I I , D. (1969) B a c t e r i p l . Rev. 33, 264-301. (2) LapidPt, Y. and de Grppt, N. (1972) Prpgr. Nucl. Acid Res. Mol. B i c l . 12, 189-228. (3) L o f t f i e l d , R. 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