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A conformational study of 5-fluorouracil labeled Escherichia coli 5SrRNA Smith, James Lewis 1980

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A CONFORMATIONAL STUDY OF 5-FLU0R0URACIL LABELED ESCHERICHIA COLI 5SrRNA by JAMES LEWIS SMITH B.Sc. U n i v e r s i t y of Puget Sound, 1971 M.Sc. U n i v e r s i t y of Puget Sound, 1974 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF.GRADUATE STUDIES i n the department of CHEMISTRY We accept t h i s t h e s i s as conforming to the re q u i r e d standard THE UNIVERSITY OF BRITISH COLUMBIA January, 1980 © James Lewis Smith In presenting this thesis in partial fulf i lment of the requirements for an advanced degree at the University of Brit ish Columbia, I agree that the Library shall make i t freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the Head of my Department or by his representatives. I t is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of Chemistry  The University of Brit ish Columbia 2075 wesbrook Place Vancouver, Canada V6T 1W5 Date December 5. 1979 DE-6 B P 75-51 1 E - i i -Supervisor: Dr. Alan G. Marshall ABSTRACT 1 9F-nmr spectroscopy and laser Raman spectroscopy are used to interpret conformational properties of 5SrRNA. When 5-fluorouracil i s added to a medium containing a c t i v e l y growing E. c o l i c e l l s i t is incorporated into the 5SrRNA (FU-5SrRNA) of the. bacteria. The 1 9F-nmr spectra of FU-5SrRNA, at 94.1 MHz and 254 MHz, are presented. They cover a chemical s h i f t range of approximately 8 p.p.m.. At 254 MHz the spectrum consists of 8 peaks and 2 shoulders, representing fluorine resonances from approximately twenty 5-fluorouracil residues in FU-5SrRNA. The most exposed residues have been assigned by comparison of the resonance frequency of the 5-fluoro-2'-deoxyuridine monophosphate monomer with that of the heat denatured FU-5SrRNA. The remainder of the fluorine resonances (about 70% of the total) are believed to be due to buried 5-fluorouracil residues. A l l the T^ values of the individual peaks were approximately the same (between 0.3 and 0.4 seconds). This i s in contrast to the T^ value of the 5-fluoro-2 *-deoxyuridine monomer which i s approximately 5 seconds. A nuclear Overhauser enhancement experiment confirms that these residues are r i g i d l y situated within the FU-5SrRNA molecule and that their molecular co r r e l a t i o n time (T) must be the same as the o v e r a l l correlation time of the macromolecule. Two important r e s u l t s are obtained from l a s e r Raman s p e c t r o s c o p i c s t u d i e s presented i n t h i s work. F i r s t , 5 - f l u o r o -s u b s t i t u t i o n a f f e c t s c e r t a i n v i b r a t i o n a l p r o p e r t i e s of the u r a c i l base. Second, from the comparison of N-5SrRNA s p e c t r a with FU-5SrRNA s p e c t r a , i t appears evident that 5 - f l u o r o u r a c i l s u b s t i t u t i o n i n 5SrRNA causes only minimal p e r t u r b a t i o n of the s t r u c t u r e ; thus, c o n c l u s i o n s r e s u l t i n g from a study of FU-5SrRNA s t r u c t u r e should be a p p l i c a b l e to N-5SrRNA. - i v -TABLE OF CONTENTS Page ABSTRACT i i TABLE OF CONTENTS i v LIST OF TABLES v i LIST OF FIGURES v i i ACKNOWLEDGEMENTS x i i CHAPTER 1. GENERAL INTRODUCTION 1 REFERENCES: CHAPTER 1 51 CHAPTER 2. EXPERIMENTAL 58 2.1. INTRODUCTION 58 2.2. BACTERIAL GROWTH 6 0 2.3. ISOLATION OF 5SrRNA 64 2.4. SEPARATION OF NORMAL 5SrRNA FROM FU-5Sr RNA 75 2.5. SAMPLE PURITY BY POLYACRYLAMIDE GEL ELECTROPHORESIS 8 0 2.6. VERIFICATION OF 5-FU INCORPORATION INTO E. COLI RNA 8 3 2.7. 1 9 F - F T NMR SPECTROSCOPY OF FU-5SrRNA 9 0 2.8. LASER RAMAN SPECTROSCOPY OF 5SrRNA AND FU—5SrRNA 103 REFERENCES: CHAPTER 2 115 CHAPTER 3. FLUORINE NUCLEAR MAGNETIC RESONANCE SPECTROSCOPY OF E. COLI FU-5SrRNA 116 REFERENCES: CHAPTER 3 141 - v -Page CHAPTER 4. LASER RAMAN SPECTROSCOPY OF N-5SrRNA AND FU-5SrRNA . 143 4.1. INTRODUCTION 143 4.2. LASER RAMAN STUDY OF 5-FU, 5-FLUORO-2 1-DEOXYURIDINE, AND 5-FLUORO-2'-DEOXYURIDINE MONOPHOSPHATE 145 4.3. LASER RAMAN SPECTROSCOPY OF N-5SrRNA AND FU-5SrRNA 156 REFERENCES: CHAPTER 4 163 CHAPTER 5. CONCLUDING REMARKS 165 REFERENCES: CHAPTER 5 168 - v i -LIST OF TABLES Table T i t l e Page 1.1 The approximate chemical s h i f t range and r e l a t i v e s i g n a l s e n s i t i v i t y , at constant f i e l d , of hydrogen, phosphorus n u c l e i 2 1.2 Estimates o f base p a i r i n g i n E. c o l i 5SrRNA obtained by o p t i c a l spectroscopy 40 2.1 The chemical composition of one l i t e r of minimal media 60 2.2 The fermentation d e v i c e s used d u r i n g v a r i o u s stages of t h i s work 61 2.3 The peak p o s i t i o n s measured i n p.p.m. r e l a t i v e to 5-FU f o r the H2O and D2O 1 9F-nmr s p e c t r a of FU-5SrRNA obtained at 254 MHz 100 2.4 Tj_ det e r m i n a t i o n s f o r the i n d i v i d u a l peaks of the D 20 sample of FU-5SrRNA at 254 MHz 102 2.5 Line i n t e n s i t i e s of the l a s e r Raman s p e c t r a shown i n F i g u r e s 2.25 and 2.26, 114 3.1 The estimated T]_ valu e s f o r the 254 MHz 1 9F-nmr spectrum of FU-5SrRNA i n D2O b u f f e r 121 3.2 The resonance frequency of the proton and the f l u o r i n e nucleus at the three most commonly employed magnetic f i e l d s t r e n g t h s ... 124 3.3 Minimum T i v a l u e s , corresponding T 2 v a l u e s , the i_ a s s o c i a t e d with divergence of and T 2 , and x i a s s o c i a t e d with the minimum f o r F i g u r e s 3.2 - 3.4 129 - v i i -LIST OF FIGURES F i g u r e T i t l e Page 1.1 A 400 megahertz low f i e l d 'H-nmr spectrum, of tRNA^ 4 1.2 The h i g h - f i e l d p r o t o n resonances o f y e a s t tRNA p^e o b t a i n e d a t s e v e r a l t e m p e r a t u r e s on a 270 megahertz FT 'H-nrar s p e c t r o m e t e r .... 5 Phe 1.3 A 3 1 p - F T nmr spectrum o f y e a s t tRNA and E. c o l i t R N A G l u a t 109 megahertz 6 1.4 The 1 9F-FT-nmr s p e c t r a of f l u o r o t y r o s i n e l a b e l e d a l k a l i n e phosphatase o f 94 mega-h e r t z and 235 megahertz 8 1.5 The c h e m i c a l s t r u c t u r e o f 5 - f l u o r o u r a c i l and 5 - f l u o r o u r i d i n e 11 1.6 The r i b o s e - p h o s p h a t e backbone s t r u c t u r e o f an RNA m o l e c u l e 14 1.7 The d i r e c t i o n a l hydrogen bonding p r o p e r t i e s o f the t h r e e most s t a b l e base p a i r s found i n RNA m o l e c u l e s 15 1.8 The g e n e r a l i z e d c l o v e r l e a f secondary s t r u c t u r e o f a l l sequenced tRNA mole-c u l e s e x c e p t f o r i n i t i a t o r tRNAs 16 1.9 A s c h e m a t i c diagram o f the c r y s t a l s t r u c t u r e o f y e a s t t R N A p h e 18 1.10 The f i r s t sequence of E. c o l i 5SrRNA 22 1.11 The c o n s t i t u e n t p a r t s o f an E. c o l i ribosome 23 1.12 The f o r m a t i o n o f the i n i t i a t i o n complex and the f u l l y f u n c t i o n a l 70S ribosome o f E. c o l i 24 1.13 P r o t e i n e l o n g a t i o n and the p e p t i d y l t r a n s f e r a s e r e a c t i o n 26 - v i i i -F i g u r e T i t l e Page 1.14 A proposed model f o r the f u n c t i o n of 5SrRNA 37 1.15 A summary of the regions of 5SrRNA which are most a c c e s s i b l e t o enzymes and chemical modifying agents . . . . 43 1.16 Two proposed models of the secondary s t r u c t u r e of E. c o l i 5SrRNA which best f i t the experimental data 45 1.17 A 300 megahertz low f i e l d 'H-nnir s p e c t r a of E. c o l i 5SrRNA at v a r i o u s temperatures .... 48 1.18 The 1 3C-nmr spectrum of the C-4 u r i d i n e carbons of Salmonella typhimurium 5SrRNA at 37°C and 75°C 49 1.19 A l a s e r Raman spectrum of a 5% aqueous s o l u t i o n of E. c o l i 5SrRNA 50 2.1 B a c t e r i a l growth curves of E. c o l i B grown on minimal media 62 2.2 A complete e l u t i o n p r o f i l e o f sRNA a p p l i e d to a Sephadex G-100 column 69 2.3 The e l u t i o n p r o f i l e f o r sRNA from a p p r o x i -mately 25 grams of E. c o l i B c e l l s from a Sephadex G-75 column 71 2.4 An e l u t i o n p r o f i l e of the 5SrRNA component obtained from F i g u r e 2.3 72 2.5 A demonstration of 5SrRNA homogeneity ac c o r d i n g to Sephadex G-75 chromatography .... 73 2.6 A schematic sid e view of the c y c l i n d r i c a l r e s e r v o i r s used to generate concave upward g r a d i e n t s of i n c r e a s i n g s a l t con-c e n t r a t i o n 77 2.7 The s e p a r a t i o n of N-5SrRNA from FU-5SrRNA by D E A E - c e l l u l o s e chromatography 78 2.8 Sample p u r i t y a c c o r d i n g to 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 82 - i x -F i g u r e T i t l e Page 2.9 A c a l i b r a t i o n c u r v e o f c o u n t i n g e f f i c i e n c y v e r s u s c h a n n e l - r a t i o s f o r samples w i t h known D.P.M. v a l u e s 85 2.10 A Sephadex G-100 chromatography o f sRNA which c o n t a i n s lhC l a b e l e d 5 - f l u o r o u r a c i l r e s i d u e s 86 2.11 A D E A E - c e l l u l o s e s a l t g r a d i e n t o f the tRNA f r a c t i o n a t e d i n F i g u r e 2.10 88 2.12 The FT 1 9F-nmr s p e c t r a o f u n f r a c t i o n a t e d n a t i v e and h e a t - d e n a t u r e d 5 - f l u o r o u r a c i l tRNA 91 2.13 The FT 1 9F-nmr s p e c t r a o f 5 - f l u o r o u r a c i l , 5 - f l u o r o - 2 ' - d e o x y u r i d i n e , FU-5SrRNA (35°C), and FU-5SrRNA (72°C) 93 2.14 The FT 1 9F-nmr spectrum o f FU-5SrRNA i n b u f f e r c o n t a i n i n g no monovalent or d i v a l e n t c a t i o n s 94 2.15 The Ti d e t e r m i n a t i o n o f t h e r m a l l y d e n a t u r e d FU-5SrRNA employing a 90°, x, 90° p u l s e t e c h n i q u e 95 2.16 The FT 1 9F-nmr spectrum o f FU-5SrRNA i n H2O b u f f e r a t 254 MHz 97 2.17 The FT 1 9F-nmr spectrum o f FU-5SrRNA i n D 20 b u f f e r a t 254 MHz 98 2.18 The T i d e t e r m i n a t i o n o f i n d i v i d u a l peaks of FU-5SrRNA ( i n D 20 b u f f e r ) a t 254 MHz 99 2.19 A n u c l e a r Overhauser enhancement e x p e r i -ment o f FU-5SrRNA ( i n D~0 b u f f e r ) a t 254 MHz 7 101 2.20 L a s e r Raman s p e c t r a o f (1) p o l y c r y s t a l l i n e 5 - f l u o r o u r a c i l , (b) p o l y c r y s t a l l i n e u r a c i l , and (c) 20 mM u r a c i l ( n e u t r a l form) i n ... 105 2.21 L a s e r Raman s p e c t r a o f 50 mM 5 - f l u o r o u r a c i l w i t h 50 mM NaC10„ i n H o0 a t d i f f e r e n t pH 106 - x -F i g u r e T i t l e Page 2.22 La s e r Raman s p e c t r a o f n e u t r a l and a n i o n i c forms o f 2 1 - d e o x y u r i d i n e i n H 20 c o n t a i n i n g 50 mM NaClC> 4 107 2.23 L a s e r Raman s p e c t r a o f n e u t r a l and a n i o n i c forms o f 2 1 - d e o x y u r i d i n e and 5 - f l u o r o - 2 1 - d e o x y u r i d i n e i n D~0 con-t a i n i n g 50 mM NaC10 4 108 2.24 Las e r Raman s p e c t r a o f n e u t r a l and a n i o n i c forms of 5 - f l u o r o - 2 ' - d e o x y u r i d i n e and 5 - f l u o r o - 2 1 - d e o x y u r i d i n e monophosphate i n H 20 c o n t a i n i n g 50 mM NaC10 4 109 2.25 Raman s p e c t r a o f aqueous samples o f N-5SrRNA b e f o r e and a f t e r d i a l y s i s 112 2.26 Raman s p e c t r a o f aqueous samples o f N-5SrRNA a f t e r d i a l y s i s and FU-5SrRNA 113 3.1 D e t e r m i n a t i o n o f T 1 by 180°, T, 90° sequences 120 3.2 The i n t r a m o l e c u l a r d i s t a n c e between f l u o r i n e and the n e a r e s t p r o t o n o f 5 - f l u o r o u r a c i l 123 —fi —ft 3.3 L o t p l o t o f T i x r and T 2 x r v e r s u s the l o g o f the o v e r a l l m o l e c u l a r c o r r e l a t i o n time (T ) a t 94.1 MHz 126 — 6 - 6 3.4 Log p l o t s o f T i x r and T 2 x r v e r s u s l o g x c c a l c u l a t e d a t 254 MHz 127 3.5 Log p l o t s o f T-^  x r ^ and T 2 x r ^ v e r s u s l o g T c c a l c u l a t e d a t 338 .7 MHz 128 3.6 An energy l e v e l diagram o f a system c o n s i s t i n g of two n u c l e a r s p i n s , I and S 132 3.7 L o g - l o g p l o t o f t r a n s i t i o n r a t e s v e r s u s r o t a t i o n a l c o r r e l a t i o n time f o r a system c o n s i s t i n g o f two u n l i k e s p i n s 136 3.8 F l u o r i n e - p r o t o n f r a c t i o n a l n u c l e a r Over-hauser enhancement f a c t o r , f j ( S ) , v e r s u s r o t a t i o n a l c o r r e l a t i o n time ( l o g s c a l e ) f o r 5 - f l u o r o u r a c i l , computed from the t r a n s i -t i o n r a t e s o f F i g u r e 3 .7 138 - x i -F i g u r e T i t l e Page 4.1 A g e n e r a l i z e d s t r u c t u r e f o r u r a c i l , 2'-deoxyuridine, 5 - f l u o r o u a c i l , 5 - f l u o r o - 2 ' - d e o x y u r i d i n e and 5 - f l u o r o -2'-deoxyuridine monophosphate 146 4.2 Laser Raman ca r b o n y l s t r e t c h i n g r e gion fo r 50 m i l l i m o l a r D2O s o l u t i o n s of n e u t r a l and a n i o n i c forms of 2'-deoxyuridine and 5 - f l u o r o - 2 ' -d e o x y u r i d i n e >. . • 149 4.3 The p r i n c i p l e resonance s t r u c t u r e s f o r n e u t r a l and a n i o n i c forms of 2'-deoxyuridine and 5 - f l u o r o - 2 ' -deoxyuridine 151 5.1 Simulated i n f r a r e d s p e c t r a f o r E. c o l i 5SrRNA s t r u c t u r a l models (—) i n comparison to the experimental spectrum recorded at 52°C (...) 168 5.2 Simulated i n f r a r e d s p e c t r a f o r E. c o l i 5SrRNA s t r u c t u r a l models (—) i n comparison to the experimental spectrum recorded at 52°C (...) 169 5.3 Simulated i n f r a r e d s p e c t r a f o r E. c o l i 5SrRNA s t r u c t u r a l models (—) i n comparison to the experimental spectrum recorded at 52°C (...) 170 5.4 Simulated i n f r a r e d s p e c t r a f o r E. c o l i 5SrRNA s t r u c t u r a l models (—) i n comparison to the experimental spectrum recorded at 52°C (...) 171 5.5 Simulated i n f r a r e d s p e c t r a f o r those E. c o l i 5SrRNA s t r u c t u r a l models in which t e r t i a r y i n t e r a c t i o n s have been proposed (—) i n comparison to the experimental spectrum recorded at 20°C ( ) 172 - x i i -ACKNOWLEDGEMENTS F i r s t of a l l I would l i k e to thank my re s e a r c h s u p e r v i s o r , Alan M a r s h a l l , f o r h i s guidance and d i r e c t i o n d u r i n g my f i v e years of a s s o c i a t i o n with h i s b i o p h y s i c a l chemistry r e s e a r c h group. I would a l s o l i k e to thank the other members of h i s group which i n c l u d e s C h r i s Roe, Greg Luoma, Bob Bruce, Junko Crothers and K.M. Lee. During v a r i o u s stages of t h i s work equipment from other departments was employed. In p a r t i c u l a r I would l i k e to thank the U n i v e r s i t y of B r i t i s h Columbia M i c r o b i o l o g y and Food Science departments, and the U n i v e r s i t y of Washington Biochem-i s t r y Department for the use of t h e i r fermentation d e v i c e s . L a s t of a l l , I would l i k e to thank c e r t a i n i n d i v i d u a l s who have been of a s s i s t a n c e during v a r i o u s stages of t h i s work. They include, Ivan Kaiser (University of Wyoming), Brian Sykes (Univer-si ty of Alberta), and Gordon Tener (University of Bri t ish Columbia). - 1 -CHAPTER 1 GENERAL INTRODUCTION B i o p h y s i c a l chemistry uses p h y s i c a l and chemical laws to i n t e r p r e t s t r u c t u r a l and f u n c t i o n a l p r o p e r t i e s of b i o l o g i c a l molecules. During the l a s t two decades X-ray c r y s t a l l o g r a p h y has provided the atomic s p a t i a l arrangement of numerous mole-c u l e s with s p e c i f i c b i o l o g i c a l f u n c t i o n s . X-ray s t r u c t u r a l a n a l y s i s has two major l i m i t a t i o n s . F i r s t , h i g h l y ordered s i n g l e c r y s t a l s of b i o l o g i c a l molecules are o f t e n d i f f i c u l t or impossible to o b t a i n . Secondly, the severe c o n s t r a i n t s on molecular motion i n a r i g i d c r y s t a l l a t t i c e are u n l i k e the n a t i v e aqueous environment where b i o l o g i c a l molecules f u n c t i o n . These two l i m i t a t i o n s have prompted i n v e s t i g a t o r s to employ p h y s i c a l methods able to provide dynamical i n f o r m a t i o n about b i o l o g i c a l molecules i n an aqueous environment. T h i s t h e s i s uses two methods, nuclear magnetic resonance (nmr) spectroscopy and l a s e r Raman spectroscopy to i n v e s t i g a t e the conformation of a s p e c i f i c r i b o n u c l e i c a c i d (RNA^ molecule i n s o l u t i o n . Hydrogen, phosphorus, and f l u o r i n e a l l have s p i n h n u c l e i which are 100% n a t u r a l l y abundant. T h e i r r e l a t i v e s e n s i t i v i t i e s and approximate chemical s h i f t ranges are given i n Table 1. Both hydrogen and f l u o r i n e have s u b s t a n t i a l s i g n a l s e n s i t i v i t y . Phosphorus has much reduced s e n s i t i v i t y but l i k e f l u o r i n e i t s - 2 -Nucleus R e l a t i v e s i g n a l s e n s i t i v i t y at c onstant f i e l d Approximate c h e m i c a l s h i f t range i n p a r t s per m i l l i o n (p.p.m.) Hydrogen F l u o r i n e 100 12 8 3 . 3 500 Phosphorus 6 . 6 3 700 Table 1.1. The approximate c h e m i c a l s h i f t range and r e l a t i v e s i g n a l s e n s i t i v i t y , a t c o n s t a n t f i e l d , o f hydrogen, f l u o r i n e and phosphorus n u c l e i ( 1 ) . c h e m i c a l s h i f t range i s l a r g e compared t o the p r o t o n . An RNA m o l e c u l e c o n t a i n s many phosphorus and hydrogen n u c l e i . How-e v e r , phosphorus-nmr ( 3 1E-nmr) and proton-nmr ('H-nrnr) are o f l i m i t e d u t i l i t y . T h i s i s due m o s t l y t o the l a r g e numbers of these n u c l e i ( i . e . 5SrRNA has 1301 hydrogen atoms and 120 phos-phorus atoms). In aqueous s o l u t i o n most of the p r o t o n r e s o n -ances are masked by the l a r g e number o f p r o t o n resonances from the w a t e r . Even i n d e u t e r i u m o x i d e the s m a l l c h e m i c a l s h i f t range of the p r o t o n resonances combined w i t h the l a r g e number of r e s o n a n c e s , due e x c l u s i v e l y t o RNA, r e s u l t i n a s p e c t r a l envelope o f u n r e s o l v a b l e peaks. There are two s m a l l p r o t o n p o p u l a t i o n s i n RNA m o l e c u l e s whose c h e m i c a l s h i f t s are f a r enough from the m a j o r i t y o f the p r o t o n resonances t h a t they are a b l e t o p r o v i d e c o n f o r m a t i o n a l i n f o r m a t i o n about these m o l e c u l e s . They can be r e s o l v e d through the a p p l i c a t i o n o f F o u r i e r Transform nmr (FT nmr) and c o r r e l a t i v e 'H-nmr t e c h -n i q u e s . One group, the exchangeable r i n g NH p r o t o n s , e l i c i t hydrogen bonding between RNA base p a i r s (see F i g u r e 1.7). - 3 -These resonances are s h i f t e d d o w n f i e l d , r e l a t i v e to the proton resonances o f water, due to r i n g c u r r e n t e f f e c t s from the h e t e r -o c y c l i c bases i n v o l v e d i n the base p a i r . The same hydrogen bonded proton resonances are i n turn s e n s i t i v e to r i n g c u r r e n t e f f e c t s from the base p a i r s which are stacked above and below them i n h e l i c a l r egions of the RNA molecules ( 2 ) . The magni-tudes of these l a t t e r e f f e c t s are of s u f f i c i e n t v a r i e t y t h a t w e l l r e s o l v e d r i n g NH hydrogen bonded proton resonances are spread out over a range of about 7 p.p.m.. A low f i e l d 'H-nmr spectrum of a v a l i n e a c c e p t i n g t r a n s f e r RNA (tRNA^3"*") from E s c h e r i c h i a c o l i (E. c o l i ) i s shown i n F i g u r e 1.1. The h e l i c a l r e g i o n s o f RNA molecules are e s s e n t i a l l y hydrophobic. Within these r e gions the exchangeable NH protons are not a c c e s s i b l e to the e x t e r n a l aqueous environment. At e l e v a t e d temperatures RNA molecules u n r a v e l exposing the hydrogen bonded protons. The r e s u l t i n g f a s t exchange of these protons with the aqueous environment causes broadening and eve n t u a l disappearance o f the resonances. T h i s technique has been used to eva l u a t e the s t a b i l i t y of h e l i c a l stem regions i n s p e c i f i c tRNA molecules ( 4 ) . I t i s a l s o a p p l i c a b l e to other types of experiments where co n f o r m a t i o n a l change a f f e c t s base s t a c k i n g i n t e r a c t i o n s ( 3 ) . The second group of proton resonances, the nonexchangeable methyl protons o f m o d i f i e d n u c l e o s i d e s , are a l s o r e s o l v a b l e using high r e s o l u t i o n ^-nmr techniques. The proton resonances, T 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 i 1 1 1 1 1 1 1 1 r 15 14 13 12 11 < 10 9 PRM.(relative to DSS) F i g u r e ^ l . l . The 400 megahertz low f i e l d 'H-nmr spectrum of tRNAj was obtained on the departmental Briiker HW 400 FT nmr spectrometer by G. Luoma and A. M a r s h a l l of t h i s l a b o r a t o r y . The RNA sample was k i n d l y provided by B. Reid (3). I t con-s i s t e d of 1 m i l l i m o l a r tRNA d i s s o l v e d i n an aqueous 10 m i l l i -molar cacodylate b u f f e r (pH 7) c o n t a i n i n g 5 m i l l i m o l a r magnesium c h l o r i d e and 1 m i l l i m o l a r EDTA. The spectrum was obtained on s i n g l e pulse c o r r e l a t i o n mode at zero dB. I t c o n s i s t s of 1000 t r a n s i e n t s at an a c q u i s i t i o n time of 0.82 seconds. The t o t a l accumulation times was approximately 11 minutes. shown i n Fig u r e 1.2, are s h i f t e d u p f i e l d r e l a t i v e to most. ' The temperature dependence of t h e i r chemical s h i f t s has been used to a s c e r t a i n the order i n which the l o c a l regions c o n t a i n i n g the modified bases u n f o l d (5-7). The dete r m i n a t i o n of l i n e widths Chemical Shift [ppm.DSS] F i g u r e 1.2. The h i g h - f i e l d proton resonances of yeast tRNA were obtained at s e v e r a l temperatures on a 270 megahertz 1H-FT nmr spectrometer. The sample c o n c e n t r a t i o n was approximately 0.55 m i l l i m o l a r . The deuterium oxide b u f f e r was 0.01 molar potassium phosphate (pH 6.6) and contained 0.1 molar KC1 and '0.01 molar MgCl 9. - 6 -and d i r e c t measurement of s p i n - l a t t i c e r e l a x a t i o n times are a l s o p o t e n t i a l l y u s e f u l f o r the i n t e r p r e t a t i o n of t h e i r motional freedom i n v a r i o u s p a r t s of the RNA molecule (8). Phosphorus, u n l i k e the proton, has a p o t e n t i a l l y l a r g e chemical s h i f t range, but i n RNA molecules most of these resonances appear to e x p e r i -ence comparable environmental e f f e c t s (9-11). T h i s i s apparent from Figure 1.3. Those resonances which are d i s p l a c e d from the m a j o r i t y are unassignable even though they do appear to be a f f e c t e d by temperature and i o n i c s t r e n g t h (12). i | r — i 1 1 1 1 1 1 r Figure 1.3. A 3 1 p - F T nmr spectrum of yeast tRNA and E. c o l i tRNA u at 109 megahertz. The water b u f f e r c o n s i s t e d of 0.01 molar sodium ca c o d y l a t e (pH 7) plus 0.1 molar sodium c h l o r i d e . The tRNAp, u sample a l s o contained 15 m i l l i m o l a r magnesium while the tRNA e sample contained about 3.5 m i l l i m o l a r magnesium. There was about 25 m i l l i g r a m s of RNA i n 0.7 m i l l i l i t e r s of buf-fer which corresponds to approximately a 1.4 m i l l i m o l a r RNA s o l u t i o n . The accumulation time f o r each spectrum was about 10 hours (9) . - 7 -The major d i s a d v a n t a g e o f both 'H-nnir and 3 1P-nmr s p e c t r o -scopy i s the presence o f a l a r g e p o p u l a t i o n o f u n a s s i g n a b l e resonances. The e x t r e m e l y u s e f u l a s p e c t o f 'H-nnir i s the con-s i d e r a t i o n o f a r e l a t i v e l y s m a l l p o p u l a t i o n o f p o t e n t i a l l y a s s i g n a b l e and i n t e r p r e t a b l e r e s o n a n c e s . T h i s o b s e r v a t i o n l e d to the f o r m u l a t i o n o f the f i r s t t e c h n i q u e employed i n t h i s t h e s i s , f l u o r i n e - n m r ( 1 9F-nmr) s p e c t r o s c o p y . The i r i v i v o i n -c o r p o r a t i o n o f a s m a l l number o f f l u o r i n e atoms a t s p e c i f i c s i t e s a l o n g the sequence o f an RNA m o l e c u l e p r o v i d e s a s m a l l p o p u l a t i o n o f s p i n % n u c l e i i n d i f f e r e n t p a r t s o f the m o l e c u l e . F l u o r i n e has o n l y s l i g h t l y l e s s s e n s i t i v i t y than the p r o t o n (83%) and a much l a r g e r c h e m i c a l s h i f t range (about 41.5 times the p r o t o n ' s ) . T h e r e f o r e , e x t e n s i v e r e s o l u t i o n o f i n d i v i d u a l resonances was e x p e c t e d . The i n t e r p r e t a t i o n o f these r e s o n -ances would p r o v i d e i n f o r m a t i o n about the l o c a l environment s u r r o u n d i n g the i n d i v i d u a l f l u o r i n e atoms l o c a t e d i n d i f f e r e n t p a r t s o f the RNA m o l e c u l e . The i r i v i v o i n c o r p o r a t i o n of f l u o r i n e i n t o a b i o l o g i c a l m o l e c u l e and subsequent 1 9F-nmr s p e c t r o s c o p y was a c h i e v e d i n 1974 ( 1 3 ) . E x p e r i m e n t o r s succeeded i n i n c o r p o r a t i n g m - f l u o r o -t y r o s i n e i n t o the t y r o s i n e s i t e s o f the d i m e r i c z i n c c o n t a i n -i n g m e t a l l o p r o t e i n , a l k a l i n e phosphatase, from E. c o l i . T h i s was a c c o m p l i s h e d by growing a t y r o s i n e a u x o t r o p h o f E. c o l i on a medium c o n t a i n i n g m - f l u o r o t y r o s i n e . The r e s u l t i n g 1 9F-nmr s p e c t r a , a t 94 megahertz and 235 megahertz, are shown i n F i g u r e 1.4. D e n a t u r a t i o n o f t h i s enzymes w i t h 6 molar g u a n i d i n e ' H C l - 8 -* 1 1 1 r — f 5 Figure 1.4. The 1 9 F - F T nmr s p e c t r a of f l u o r o t y r o s i n e l a b e l e d a l k a l i n e phosphatase at 94 megahertz and 235 megahertz. The aqueous b u f f e r c o n s i s t e d of 0.1 molar T r i s (pH 7.9) and the p r o t e i n c o n c e n t r a t i o n was 0.30 m i l l i m o l a r . The accumulation time for these s p e c t r a was about 30 minutes (14). - 9 -y i e l d e d a s i n g l e f l u o r i n e resonance at approximately 58.8 p.p.m. (15). Comparison of t h i s spectrum with those of the n a t i v e enzyme, shown above, suggest t h a t resonances 7-10 are the most n e a r l y exposed to the s u r f a c e environment of the pro-t e i n . Resonances 1-6 show monotonic i n c r e a s e s i n l i n e - w i d t h with i n c r e a s e d chemical s h i f t to lower f i e l d . T h i s i s due to the b u r i e d m - f l u o r o t y r o s i n e s which experience t e r t i a r y i n t e r -a c t i o n s with the surrounding p r o t e i n environment. The data from t h i s 1 9F-nmr study was compared with t h e o r e t i c a l models in an e f f o r t to determine the e f f e c t of the p r o t e i n environment upon these f l u o r i n e probes. For example, c a l c u l a t i o n s of s p i n -l a t t i c e r e l a x a t i o n times ( T ^ ) , at 94 megahertz, assuming only d i p o l e - d i p o l e r e l a x a t i o n v i a the t r y o s i n e r i n g proton nearest the f l u o r i n e atom provided an absolute minimum value of 0.45 seconds (16). Comparison with experimental values of , f o r each resonance, i n d i c a t e d t h at a l l except resonance 10 are sub-s t a n t i a l l y l e s s than the t h e o r e t i c a l minimum v a l u e . T h i s im-p l i e d t h a t an i n t e r m o l e c u l a r d i p o l a r r e l a x a t i o n mechanism e x i s t e d t h at i n v o l v e d the f l u o r i n e atom and the protons of the surrounding p r o t e i n environment. T h e i r d i s t a n c e s from the f l u o r i n e atom must be comparable to that of the v i c i n a l proton on the t y r o s i n e r i n g . Using a s i m i l a r s t r a t e g y experimental r e s u l t s were combined with t h e o r e t i c a l c o n s i d e r a t i o n s to pro-vide d e f i n i t i v e i n f o r m a t i o n about the molecular dynamics of m - f l u o r o t y r o s i n e a l k a l i n e phosphatase from E. c o l i . They have examined the e f f e c t s of i n c r e a s i n g magnetic f i e l d s t r e n g t h on - 10 -chemical s h i f t a n i s o t r o p y , the degree of i n t e r n a l m o b i l i t y f o r i n d i v i d u a l m - f l u o r o t y r o s i n e s and a molecular c o r r e l a t i o n time for the enzyme which i s c o n s i s t e n t with f l u o r e s c e n c e d e p o l a r -i z a t i o n data (15). A l s o c o n s i d e r e d were induced c o n f o r m a t i o n a l changes i n the enzyme through z i n c or phosphate b i n d i n g and t h e i r e f f e c t s on the 1 9F-nmr spectrum (17). One of the major goals of t h i s t h e s i s was to c o n s i d e r the a p p l i c a b i l i t y of the same type of study to a s p e c i f i c RNA molecule of b i o l o g i c a l i n t e r e s t . These c o n s i d e r a t i o n s w i l l be d e a l t with e x t e n s i v e l y i n Chapter 3. The a c t i o n of the a n t i c a r c i n o g e n 5 - f l u o r o u r a c i l (5-FU) on E. c o l i suggested that an experiment analogous to the m-fluoro-t y r o s i n e l a b e l e d a l k a l i n e phosphatase study was f e a s i b l e (18-19). The drug, shown i n F i g u r e 1.5, w i l l i n c o r p o r a t e i n t o the bac-t e r i a l RNA i f i t i s added duri n g e a r l y e x p o n e n t i a l growth. The i n c o r p o r a t i o n i s s p e c i f i c f o r s i t e s normally c o n t a i n i n g u r i d i n e or u r i d i n e - d e r i v e d bases. The o n l y known study concerning the Va 1 f u n c t i o n a l i t y of a s p e c i f i c 5-FU c o n t a i n i n g tRNA (FU-tRNA ) i n d i c a t e s t h at replacement of 95% of the u r i d i n e and u r i d i n e -d e r i v e d bases by 5-FU d i d not a p p r e c i a b l y a l t e r i t s a b i l i t y to become amino a c y l a t e d (20). An important c o n s i d e r a t i o n f o r t h i s study of f l u o r i n e con-t a i n i n g n u c l e i c a c i d s : w i t h 1 9F-nmr was the n e c e s s i t y f o r s u f -f i c i e n t l y l a r g e sample q u a n t i t i e s to overcome the i n h e r e n t low s e n s i t i v i t y of nmr spectroscopy. The study of a s p e c i f i c FU-tRNA would have had d i s t i n c t advantages. I t i s r e l a t i v e l y - 11 -o F i g u r e 1.5. The chemical s t r u c t u r e of 5 - f l u o r o u r a c i l (R=H) and 5 - f l u o r o u r i d i n e (R=ribose s u g a r ) . sma l l i n s i z e , has a known secondary s t r u c t u r e , and a ra t h e r w e l l understood f u n c t i o n i n the c e l l . However, the requirement of l a r g e sample amounts f o r a s u f f i c i e n t s i g n a l to noise r a t i o on the department V a r i a n FT-XL100 l e d to the s e l e c t i o n of 5S ribosomal RNA (5SrRNA) f o r t h i s study. I t r e p r e s e n t s about 3% of the t o t a l c e l l u l a r RNA of E. c o l i while a s p e c i f i c tRNA accounts f o r on l y about 0.8% of the t o t a l (21). The primary o b j e c t i v e of t h i s 1 9F-nmr study of 5-FU con-t a i n i n g 5SrRNA ( FU-5SrRNA) from E. c o l i was to observe the - 12 -i n d i v i d u a l f l u o r i n e resonances and i n t e r p r e t the e f f e c t s of a n u c l e i c a c i d environment upon them. Then to induce conforma-t i o n a l changes i n the molecule and observe the e f f e c t s on the f l u o r i n e resonances as the immediate chemical environment about each of the molecular probes changed with a l t e r a t i o n s i n macro-molecular conformation. A major disadvantage of any study of t h i s type i s that the molecular probe i s not n a t i v e to the b i o l o g i c a l system. The c o v a l e n t l y bound f l u o r i n e atom i s s m a l l (about the s i z e of an OH group) and normally u n r e a c t i v e . However, some f l u o r i n a t e d p y r i m i d i n e s are h i g h l y t o x i c and known to markedly i n h i b i t the growth of a c t i v e l y d i v i d i n g c e l l s (19). The i n t r o d u c t i o n of a h i g h l y e l e c t r o n e g a t i v e f l u o r i n e atom at a p o s i t i o n normally occupied by a proton a l -t e r s c e r t a i n p h y s i c o c h e m i c a l p r o p e r t i e s of the v i c i n a l atoms ( i . e . pK of the N-3 proton of u r a c i l and the e x t i n c t i o n co-a e f f i c i e n t at 260 nanometers). Therefore the presence of f l u o -r i n e c o u l d p o s s i b l y a l t e r base p a i r i n g and base s t a c k i n g i n t e r -a c t i o n s i n v o l v i n g 5-FU. The concern over the p o s s i b l e e f f e c t s of 5-FU on RNA con-formation l e d to the f o r m u l a t i o n of the second p h y s i c a l method employed i n t h i s t h e s i s , l a s e r Raman spectroscopy. S p e c t r a of n a t i v e 5SrRNA and . FU-5SrRNA were obtained in order to d e t e r -mine the e f f e c t s of 5-FU i n c o r p o r a t i o n on macromolecular con-formation. The Raman spectrum of a p o l y n u c l e o t i d e i s the sum of r i n g v i b r a t i o n s from the i n d i v i d u a l nitrogenous bases p l u s v i b r a t i o n a l c o n t r i b u t i o n s due to the phosphate backbone which - 13 -l i n k s the n u c l e o t i d e s together. T h i s meant that Raman s p e c t r a of u r i d i n e and 5 - f l u o r o u r i d i n e monomers a l s o had to be com-pared before i t was p o s s i b l e to i n f e r s i m i l a r i t i e s or d i f f e r -ences i n the macromolecular conformation of 5SrRNA due to f l u o r i n e i n c o r p o r a t i o n . The u t i l i t y of l a s e r Raman s p e c t r o -scopy as a p p l i e d to n u c l e i c a c i d s i s that i n t e n s i t i e s of Raman l i n e s are s e n s i t i v e to conformation. T h i s w i l l be d i s c u s s e d e x t e n s i v e l y i n Chapter 4. RNA molecules are r i b o n u c l e o t i d e polymers of the h e t e r o -c y c l i c bases adenine (A) , guanine (G), u r a c i l (U), c y t o s i n e (C) , and a s m a l l number of m o d i f i e d bases. The primary s t r u c -ture c o n s i s t s of s p e c i f i c sequences of these bases t h a t are i n t e r c o n n e c t e d through phosphodiester l i n k a g e s between 3' and 5' hydroxyls of the r i b o s e m o i e t i e s . T h i s i s i l l u s t r a t e d i n F i g u r e 1.6. Molecules of tRNA are approximately 75-80 nucl e o -t i d e s long while 23S ribosomal RNA (23SrRNA), 16SrRNA, and 5SrRNA are about 3700 , 1700 , and 120 n u c l e o t i d e s r e s p e c t i v e l y . These molecules f o l d i n t o ^ s p e c i f i c secondary s t r u c t u r e s i n accordance with the d i r e c t i o n a l hydrogen bonding p r o p e r t i e s of the i n d i v i d u a l bases. The most s t a b l e base p a i r s i n RNA molecules are G-C, A-U, and G-U p a i r s which are shown i n F i g -ure 1.7. The s t r o n g e s t base p a i r , G-C, c o n s i s t s of three hydrogen bonds while the A-U and G-U p a i r s have onl y two hydrogen bonds. A l l of the over 87 sequenced tRNA molecules appear to have a comparable secondary s t r u c t u r e (23). T h i s s o - c a l l e d . ' " - 14 --0—P=o I 0 I H ' L ^ ° \ b a s e 0 OH - 0 — P = 0 I 0 I ^ C / O ^ i j a s e P OH Figure 1 . 6 . The ribose-phosphate backbone structure of an RNA molecule. cloverleaf structure i s shown in Figure 1 . 8 . It consists of five d i s t i n c t regions. The major function of two of these re-gions, the acceptor stem and the anticodon loop /are well es-tablished. The 3 ' terminus of the acceptor stem is the s i t e of attachment of the amino acid, while the anticodon loop i s the region where tRNA recognizes the mRNA codon, at the ribo-some, during protein synthesis. The functional roles of the - 1 5 -Figure 1.7. The d i r e c t i o n a l hydrogen bonding p r o p e r t i e s of the three most s t a b l e base p a i r s found i n RNA molecules (22). - 16 -P-o-o-I ?-?-5 O -I O -D LOOP 15 \ 3 - O - Y - O A' o— o— R-o| V A c i o i -o I - o I -fro -o i -o I -o I -o \ 651 ACCEPTOR STEM T¥C LOOP • T 6 0 .4 6—o—o-6- 6, R / " A — ANTICODON LOOP Y I U \ -o I -o I -o I "9. 4 0 -o \ o I H / - 0 — 0 35 4 5 ' VARIABLE LOOP ANTICODON Figure 1.8. The generalized cloverleaf secondary structure of a l l sequenced tRNA molecules except for i n i t i a t o r tRNAs (24). other three regions, the D loop, the variable loop, and the TTC loop, are less well understood. The T^C region may bind to 5SrRNA during the binding of tRNA to the ribosome. This w i l l be discussed in some d e t a i l subsequently. A complete t e r t i a r y structure i s available for only one Phe s p e c i f i c RNA molecule, yeast tRNA (25-26). X-ray c r y s t a l -lography, to a resolution of 2.5 angstroms, has v e r i f i e d the cloverleaf secondary structure. The secondary hydrogen bonds - 17 -are r e s p o n s i b l e f o r most of the s t r u c t u r a l i n t e g r i t y of t h i s RNA molecule. A l l secondary base p a i r s are of the G-C and A-U type except f o r a s i n g l e G-U p a i r i n the acceptor stem. The stem regions form RNA-A type r i g h t handed a n t i p a r a l l e l double h e l i c e s of about 10-12 bases per turn and have a diameter of approximately 20 angstroms. These h e l i c a l regions c o n s i s t of c l o s e l y stacked base p a i r s . The secondary hydrogen bonds wi t h -i n these regions are v i r t u a l l y i n a c c e s s i b l e to the surrounding aqueous environment. In f a c t about 90% of the bases i n yeast Phe tRNA co u l d be c l a s s i f i e d as i n t e r n a l hydrophobic (27). The acceptor stem c o n s i s t s of 7 base p a i r s and the D stem has 4 base p a i r s . Both the T^C stem and the anticodon stem have 5 base p a i r s . Besides these'secondary hydrogen bonds there are 11 t e r t i a r y base p a i r s which f o l d the D arm toward the T^C arm. Phe . A schematic diagram of the c r y s t a l s t r u c t u r e of tRNA i s shown i n F i g u r e 1.9. The molecule i s L-shaped with the a n t i -codon r e g i o n at one end and- the 3' terminus at the other end. The two p e r p e n d i c u l a r axes are each about 60 angstroms i n length and the s i t e of amino a c i d attachment (the 3' terminus) i s separated from the anticodon by approximately 80 angstroms. The 7 bases i n the acceptor stem and the 5 bases of the TTC stem form a continuous h e l i x , with one gap, along the h o r i z o n -t a l a x i s . The v e r t i c a l a x i s i s a continuous h e l i x , with two gaps, that c o n s i s t s of the 4 base p a i r s i n the D stem and the 5 base p a i r s of the anticodon stem. - 18 -T arm F i g u r e 1.9p^ e A schematic diagram of the c r y s t a l s t r u c t u r e of yeast tRNA . The base p a i r e d regions are represented as long bars while s i n g l e bases are short bars. The t e r t i a r y base p a i r s are i n d i c a t e d by s o l i d dark rods (28). Besides secondary and t e r t i a r y base p a i r s there are other Phe f a c t o r s which c o n t r i b u t e to the molecular s t a b i l i t y of tRNA They i n c l u d e d i v a l e n t c a t i o n s , t e r t i a r y hydrogen bonds i n v o l v -ing r i b o s e s and phosphates and the i o n i c s t r e n g t h of the aque-ous environment. The most important d i v a l e n t c a t i o n i s mag-nesium. Both tRNA and 5SrRNA molecules assume f u n c t i o n a l conformations i n the presence of magnesium and can e x i s t i n n o n f u n c t i o n a l conformations i n i t s absence (29-31) . The number Phe of magnesium b i n d i n g s i t e s i n tRNA has not been agreed upon; - 19 -even X-ray c r y s t a l l o g r a p h y leaves some doubts (32) . The second f a c t o r , hydrogen bonding of the r i b o s e s and the phosphates, i n v o l v e s the 2'-oxygens of the r i b o s e s and some bases as hydro-gen donors or acceptors and a phosphate oxygen as a hydrogen acceptor (33). These i n t e r a c t i o n s are much l e s s s t e r e o s p e c i f i c and not as i s o l a t e d as secondary and t e r t i a r y base p a i r s . T h e r e f o r e , they are much harder to a s s i g n . The f i n a l c o n t r i -butor to RNA s t a b i l i t y , the i o n i c s t r e n g t h of the aqueous en-vironment, i s important f o r s t a b i l i z a t i o n o f the r i b o s e phos-phate backbone. The backbone i s p o l y a n i o n i c at p h y s i o l o g i c a l pH due to a negative charge on one of the f r e e phosphate oxy-gens. The presence of p o s i t i v e monovalent c a t i o n s , such as sodium and potassium, n e u t r a l i z e l i k e charge coulombic r e p u l -s i o n between the n e g a t i v e l y charged phosphate oxygens. The three dimensional p i c t u r e provided by the X-ray s t r u c -Phe t u r a l a n a l y s i s of yeast tRNA serves as a model f o r s t r u c -t u r a l i n t e r p r e t a t i o n s of a l l RNA molecules. The same types of i n t e r a c t i o n s mentioned above c o n t r i b u t e to the molecular s t a -Phe b i l i t y of a l l RNA molecules. A s i n g l e c r y s t a l of tRNA used to o b t a i n e l e c t r o n d e n s i t y maps f o r s t r u c t u r a l a n a l y s i s i s Phe about 75% water (34). S t i l l these tRNA molecules are im-mobile and the p i c t u r e provided from X-ray s t u d i e s i s a s t a t i c one. Numerous s t u d i e s i n a more n a t i v e aqueous environment tend to s u b s t a n t i a t e s i m i l a r i t i e s between the c r y s t a l s t r u c t u r e Phe and the s o l u t i o n s t r u c t u r e of tRNA . These i n c l u d e low f i e l d 'H-nmr (35-36), high f i e l d 'H-nmr (37) , base s p e c i f i c chemical - 20 -m o d i f i c a t i o n ( 3 8 ) , s m a l l angle X-ray s c a t t e r i n g ( 3 9 ) , f l u o r e s -cence s t u d i e s ( 4 0 ) , complementary o l i g o n u c l e o t i d e b i n d i n g ( 4 1 ) , u l t r a v i o l e t i n d u ced c r o s s l i n k i n g e x p e r i m e n t s ( 4 2 ) , t r i t i u m l a b e l i n g s t u d i e s ( 4 3 ) , and enz y m a t i c a c c e s s i b i l i t y s t u d i e s ( 4 4 ) . However, tRNA m o l e c u l e s are known t o have many i n t e r a c t i o n s w i t h p r o t e i n s and o t h e r n u c l e i c a c i d s . A s p e c i f i c tRNA can be c o n s i d e r e d as a s u b s t r a t e f o r more than a dozen p r o t e i n s ( 4 5 ) . Many of these i n t e r a c t i o n s are b e l i e v e d t o cause (induce) con-f o r m a t i o n a l changes i n tRNA m o l e c u l e s which cannot be m o n i t o r e d w i t h X-ray c r y s t a l l o g r a p h i c techniques.. The u n i v e r s a l c l o v e r l e a f secondary s t r u c t u r e i s c o r r o b o r -Phe at e d by the X-ray s t r u c t u r e o f tRNA . The secondary s t r u c -t u r e s o f 5SrRNA and o t h e r l a r g e r RNA m o l e c u l e s are the s u b j e c t o f much debate. T h i s a m b i g u i t y w i t h r e g a r d t o secondary s t r u c -t u r e i s i n p a r t due t o the l a r g e s i z e s o f these RNA m o l e c u l e s . The RNA m o l e c u l e o f p r i m a r y concern i n t h i s s t u d y i s E. c o l i 5SrRNA. I t was f i r s t i s o l a t e d by two independent r e s e a r c h groups i n 1964 (46-47). A l t h o u g h i t l a c k e d m e t h y l a t e d bases and p s e u d o u r i d i n e s , i t s o v e r a l l base c o m p o s i t i o n was s i m i l a r t o tRNA. T h i s l e d one o f the groups t o the er r o n e o u s c o n c l u s i o n t h a t 5SrRNA was a p r e c u r s o r o f tRNA and t h a t i t became m e t h y l -ated a t a l a t e r stage (46). T h e i r i n a b i l i t y t o amino a c y l a t e 5SrRNA was a c l u e t h a t i t was not a tRNA p r e c u r s o r ( 4 6 ) . The o t h e r group c o r r e c t l y c o n c l u d e d t h a t 5SrRNA was a t t a c h e d t o the b a c t e r i a l ribosome and e s t i m a t e d i t s c h a i n l e n g t h t o be approx-i m a t e l y 105 n u c l e o t i d e s ( 47). - 21 -i During p r e l i m i n a r y s t u d i e s of E. c o l i 5SrRNA there was much s p e c u l a t i o n concerning i t s o r i g i n . Besides being a p o s s i b l e p recursor to tRNA, i t was suspected to be a degradation product of messenger RNA (mRNA) or the other l a r g e r rRNA molecules. The l i k e l i h o o d t h at i t was a random degradation product was excluded by i t s apparent homogeneity with r e s p e c t to ch a i n l e n g t h and a unique 3" and 5' terminus (48). In 1966, i t was confirmed that the two types of molecules, 5SrRNA and tRNA, d i d not e x h i b i t a p p r o p r i a t e sequence homologies and that the - methyl a c c e p t i n g a c t i v i t y of 5SrRNA, observed p r e v i o u s l y , was not due to 5SrRNA but contaminating submethylated tRNA attached to the ribosome (49). T h i s e l i m i n a t e d the p o s s i b i l i t y that 5SrRNA was a pre-cursor of tRNA. At about t h i s time i t became apparent that 5SrRNA was u b i q u i t o u s . Besides i t s presence i n E. c o l i , i t was found to be a s s o c i a t e d with the ribosomes of yeast (50), r a t l i v e r s (51) , sea u r c h i n s (52) and amphibians (53) . In 1968 the f i r s t sequence of E. c o l i 5SrRNA was p u b l i s h e d (54). T h i s se-quence i s shown i n Fig u r e 1.10. I t c o n s i s t e d of 120 n u c l e o t i d e s and had a molecular weight of 40,575 d a l t o n s . A l l of the n u c l e -o t i d e s were A, U, G, and C; u n l i k e tRNA no unusual bases were observed. Today the 5SrRNA sequences from a v a r i e t y of organ-isms are a v a i l a b l e (55). The major components of the E. c o l i ribosome are shown i n Figure 1.11. A 5SrRNA molecule i s an i n t e g r a l p a r t of each b a c t e r i a l ribosome. I t , along with 23SrRNA and approximately 34 p r o t e i n s , c o n s t i t u t e s the 50S ribosomal subunit of E. c o l i - 22 -o 708 O CD > C a o c 80 > n o o o o n o n o I I I I I i ! I UCCCCAUGC 90 g 100 6^ % cy 1 K D O - O 1 0 c —o C D — O CD — O CD — O O < 5 0 O a < < CD O O O 3 4 0 I I I I I ^ 3 0 GUGGUCcCT G*20 1 2 0 C o x Figure 1.10. The f i r s t sequence of E. c o l i 5SrRNA. The se-quence i s drawn with an o r i g i n a l l y proposed base p a i r i n g scheme (54). (56). The three RNA molecules combined account f o r approx-imately 63% of the ribosome's mass (56). Through c o o p e r a t i v e i n t e r a c t i o n s between these n u c l e i c a c i d s and p r o t e i n s the ribosome f u n c t i o n s as the p r o t e i n assembly apparatus of the c e l l . - 23 -SOS Figure 1.11. The c o n s t i t u e n t p a r t s of an E. c o l i ribosome. In E. c o l i , 70S ribosomes undergo c o n t i n u a l a s s o c i a t i o n and d i s s o c i a t i o n i n t o 30S and 50S subunits during p r o t e i n syn-t h e s i s (57-58). The fundamental steps i n v o l v e d i n the i n i t i -a t i o n of a f u l l y f u n c t i o n a l ribosome are o u t l i n e d i n Figure 1.12. The f i r s t step i s the d i s s o c i a t i o n of an i n a c t i v e 70S ribosome i n t o i t s two elemental s u b u n i t s . Next three i n i t i -a t i o n f a c t o r p r o t e i n s (IF-1, IF-2, and IF-3) bind to the 30S subunit i n the order shown i n Figure 1.12. The f i r s t ; to bind i s IF-3. I t i s b e l i e v e d to a c t u a l l y be two p r o t e i n s having - 24 -50S P Inactive 70S ribosome 30S subunit with bound mRNA / s o s \ 0 I subunit I GTP-(iF^)-£Met-tRNA Initiation codon Initiation complex » » { J M ) +(£2) +(£3) K G D P + P. Functional 70S ribosome F i g u r e 1.12. The formation of the i n i t i a t i o n complex and the f u l l y f u n c t i o n a l 70S ribosome of E. c o l i . IF-1, IF-2, and IF-3 are the i n i t i a t i o n f a c t o r p r o t e i n s (57). molecular weights of approximately 21,500 and 23,500 d a l t o n s (59). Formation of t h i s 30S-IF-3 complex appears to prepare - 25 -the s u b u n i t t o r e c e i v e mRNA, which ne x t b i n d s t o t h i s complex. Now I F - 1 (a s i n g l e p r o t e i n w i t h a m o l e c u l a r weight o f 9,400 d a l t o n s ) ; guanosine 5 ' - t r i p h o s p h a t e (GTP), IF-2 (two p r o t e i n s o f 80,000 and 92,000 d a l t o n s ) , and i n i t i a t o r tRNA ( N - f o r m y l -methionyl-tRNA i n E. c o l i ) b i n d t o the 30S x IF-3 x mRNA com-p l e x ( 5 9 ) . The subsequent r e a s s o c i a t i o n o f t h i s complex w i t h the 50S s u b u n i t i n d u c e s the r e l e a s e o f the t h r e e i n i t i a t i o n f a c t o r s w h i l e c o n v e r t i n g a m o l e c u l e o f GTP t o guanosine 5'-dip h o s p h a t e (GDP). Accompanying t h i s r e a s s o c i a t i o n o f the two r i b o s o m a l s u b u n i t s i s an a l i g n m e n t o f i n i t i a t o r tRNA i n t o the r i b o s o m a l P s i t e l o c a t e d on the 50S s u b u n i t . The r i b o s o m a l A s i t e , a l s o l o c a t e d on the 50S s u b u n i t , i s i n c l o s e p r o x i m i t y t o the P s i t e . These two s i t e s are i m p l i c a t e d i n p r o t e i n s y n -t h e s i s . The f i r s t s t e p , e l o n g a t i o n , i s shown i n F i g u r e 1.13. I t c o n s i s t s o f the al i g n m e n t o f an amino a c y l tRNA i n t o the A s i t e , f o l l o w e d by p e p t i d e bond f o r m a t i o n between the amino group o f the amino a c i d a t t a c h e d t o the tRNA i n the A s i t e and the c a r b o x y l group o f the i n i t i a t o r tRNA 1s amino a c i d i n the P s i t e . T h i s i s a l s o i l l u s t r a t e d i n F i g u r e 1.13. T h i s p e p t i d e bon<^ f o r m a t i o n i s accompanied by a c l e a v a g e a t the e s t e r l i n k -age h o l d i n g f - m e t h i o n i n e t o the i n i t i a t o r tRNA. The n e x t s t e p f o l l o w i n g e l o n g a t i o n i s c a l l e d t r a n s l o c a -t i o n . I t i n v o l v e s a t r a n s f e r o f the p e p t i d y l tRNA a t the A s i t e t o the P s i t e and movement o f mRNA so t h a t the next t r i p -l e t codon i s i n p r o x i m i t y t o the A s i t e . C o n c u r r e n t l y , the tRNA p r e v i o u s l y o c c u p y i n g the P s i t e i s r e l e a s e d from the - 26 -Et9nq3tJ9n Aminoacyl site (A) Peptidy! site (P) Ser Ribosomi EF-T)—GTP Incoming aminoacyl-tRNA bound to EF-T —GTP complex J - 1 — 1 U J U_ l _ (Ala) (Ser) ^ (Tyr) (Phe) \ / Codons The peptidyl transferase reaction Empty __(_Pf 5'— i - U I I I I I I L L L (fMet)f (Ala) (Ser)N(Tyr) (Phe) fMet—Ala 5' I-"-" LJ. ^,^Gj_GTP ^ •(EFj)) + GDP + 1 Ribosome is now ready for next aminoacyl-tRNA u - i • • • " (fMet) (AIa)( (Ser) (Tyr)\[Phe) Pept idvt T r a n s -f e r a s e React ion Peptidyl-tRNA I 0 = C I NH I R,— CH I C f l 5' end of mRNA Peptidyl site \ R-—CH I C II o • "^^ 2 Aminoacyl site \ New aminoacyl-tRNA 0 = C NH I • R, CH 0 = C I NH I R, CH \ Empty tRNA peptidyl transferase of 50S subunit Psite c — o -Lengthened peptidyl-tRNA LSI A site 3' end Figure 1.13. P r o t e i n e l o n g a t i o n i s shown on the l e f t . EF-T and EF-G are the e l o n g a t i o n f a c t o r s . To the r i g h t the d e t a i l s of the p e p t i d y l t r a n s f e r a s e r e a c t i o n , which occurs during e l o n g a t i o n , are shown (60). - 27 -r i b o s o m e . The A s i t e i s f r e e t o r e c e i v e t h e n e x t a mino a c y l -a t e d tRNA. T h e r e i s a t l e a s t one s p e c i f i c tRNA f o r e a c h a mino a c i d . Amino a c y l a t i o n o f t h e 3' t e r m i n u s o f tRNA i s c a t a l y z e d by s y n t h e t a s e enzymes t h a t a r e a l s o s p e c i f i c f o r e a c h tRNA s p e c i e s . The b i n d i n g o f an amino a c y l a t e d tRNA t o t h e r i b o -s o m a l A s i t e i s p r e c e d e d ^ by t h e b i n d i n g o f one o f t h e s u b u n i t s o f t h e d i m e r i c c y t o p l a s m i c p r o t e i n e l o n g a t i o n f a c t o r - T ( E F - T ) . The two s u b u n i t s a r e c a l l e d e l o n g a t i o n f a c t o r - T (EF-T ) and e l o n g a t i o n f a c t o r - T u (EF-T ) . EF-T f i r s t a s s o c i a t e s w i t h GTP c a u s i n g t h e r e l e a s e o f t h e E F - T g s u b u n i t . The GTP x EF-T^ c o m p l e x t h e n b i n d s t o t h e amino a c y l a t e d tRNA (aa-tRNA) t o f o r m a GTP x E F - T u x aa-tRNA c o m p l e x . T h i s c o m p l e x b i n d s t o t h e r i b o s o m a l A s i t e s o t h a t t h e a n t i c o d o n l o o p o f tRNA' i s s i t u -a t e d t o h y d r o g e n bond t o t h e c o m p l e m e n t a r y mRNA p r o x i m a l t o t h e A s i t e . C o m p l e m e n t a r i t y i s e s s e n t i a l f o r t h e b i n d i n g o f tRNA t o t h e A s i t e . The amino g r o u p o f t h e amino a c y l a t e d tRNA i n t h e A s i t e i s s i t u a t e d i n c l o s e p r o x i m i t y t o t h e e s t ex i f l e d c a r -b o x y l c a r b o n o f t h e amino a c y l a t e d tRNA i n t h e P s i t e . N u c l e o -p h i l i c a t t a c k by t h e amino g r o u p i s f a c i l i t a t e d by t h e enzyme p e p t i d y l t r a n s f e r a s e (see F i g u r e 1 . 1 3 ) . E l o n g a t i o n and t r a n s -l o c a t i o n r e q u i r e a p p r o x i m a t e l y 50 m i l l i s e c o n d s ( 6 1 ) . The p o l y -p e p t i d e w i l l c o n t i n u e t o grow by r e p e t i t i o n o f t h e s e two s t e p s u n t i l t h e mRNA c o d o n f o r t e r m i n a t i o n i s s i g n a l e d . I n E. c o l i t h e r e a r e t h r e e t e r m i n a t i o n c o d o n s , UAG, UAA, and UGA w h i c h do n o t have c o m p l e m e n t a r y tRNA a n t i c o d o n s . D u r i n g t e r m i n a t i o n , t h e p o l y p e p t i d y l - t R N A s h i f t s f r o m t h e A s i t e . t o t h e P s i t e . A c t u a l - 28 -t e r m i n a t i o n i s promoted by t h r e e p r o t e i n s c a l l e d r e l e a s e f a c -t o r s , R-^ , R 2/ and R^. They are a b l e t o r e c o g n i z e the t e r m i n a -t i o n codons o f mRNA (62). H y d r o l y s i s o f the e s t e r bond be-tween the p o l y p e p t i d e c h a i n and t e r m i n a l tRNA i s most l i k e l y c a t a l y z e d by p e p t i d y l t r a n s f e r a s e whose c a t a l y t i c p r o p e r t i e s are changed by the r e l e a s i n g f a c t o r s (57) . Now the l a s t tRNA and mRNA l e a v e the 70S ribosome which i n t u r n d i s s o c i a t e s i n t o 30S and 50S s u b u n i t s u n t i l another i n i t i a t o r tRNA s i g n a l s r e -f o r m a t i o n o f a f u n c t i o n a l ribosome t o c o n s t r u c t another p r o t e i n . The amino a c i d sequence o f a p r o t e i n depends upon the o r d e r i n which s p e c i f i c amino a c y l a t e d tRNA m o l e c u l e s b i n d t o the A s i t e . T h i s o r d e r i s dependent upon the sequence o f the t r i p l e t n u c l e -o t i d e codons o f mRNA. T h i s r e g i o n o f r e c o g n i t i o n i s l o c a t e d i n p r o x i m i t y t o the A s i t e and d i r e c t l y a d j a c e n t t o the p r e c e e d -i n g p e p t i d y l tRNA which o c c u p i e s the P s i t e . Each s p e c i f i c tRNA has an a n t i c o d o n l o o p c o n t a i n i n g t h r e e bases which r e c o g -n i z e s the complementary t r i p l e t codon o f mRNA. Du r i n g the e l o n g a t i o n - t r a n s l o c a t i o n p r o c e s s the mRNA a l s o moves a l o n g so t h a t a new t h r e e base codon i s s i t u a t e d i n the v i c i n i t y o f the A s i t e . The s i t e i s now ready t o r e c e i v e a new amino a c y l tRNA a c c o r d i n g t o the c o m p l e m e n t a r i t y o f the codon and a n t i c o d o n . Codon-anticodon r e c o g n i t i o n i n v o l v e s the f o r m a t i o n o f Watson-C r i c k base p a i r s . A c c o r d i n g t o the "wobble h y p o t h e s i s " the base p a i r i n g i s most s t r i n g e n t f o r the c e n t r a l o f the t h r e e bases and one o f the o u t e r bases ( 6 3 ) . The base p a i r i n g p r o p e r -t i e s o f the t h i r d base are p r o b a b l y l e s s w e l l d e f i n e d . - 29 -5SrRNA i s e s s e n t i a l f o r p r o t e i n s y n t h e s i s but i t s s p e c i f i c f u n c t i o n i s not completely understood. The.most widely accepted hypothesis i s that i t a s s i s t s i n the bi n d i n g of tRNA to the A s i t e of the ribosome through a mechanism comparable to codon-,anticodon r e c o g n i t i o n : that i s , by base p a i r i n g of a s p e c i f i c region of 5SrRNA to the complementary sequence of tRNA. The most l i k e l y candidates f o r these regions have been suggested from sequence homology s t u d i e s of 5SrRNA and tRNA from d i f f e r e n t p r o c a r y o t e s and d i f f e r e n t s t r a i n s of E. c o l i . D i f f e r e n t s t r a i n s of E. c o l i 5SrRNA e x h i b i t v a r i a b i l i t y at both the 3' and 5' ends. The middle r e g i o n , bases 14-91, i s h i g h l y conserved (64). Comparisons between the sequences of d i f f e r e n t p r o c a r y o t e s i n -d i c a t e a C-G-A-A segment near p o s i t i o n 45 (55, 65). E. c o l i 5SrRNA a l s o has the same segment about p o s i t i o n 107. This l a t -ter segment i s not l i k e l y to be f u n c t i o n a l l y important s i n c e B a c i l l u s s t e a r o t h e r m o p h i l i s (B. s t e a r o t h e r m o p h i l i s ) 50S r i b o -somal subunits r e c o n s t i t u t e d with E. c o l i 5SrRNA are b i o l o g i c a l l y a c t i v e , but B. s t e a r o t h e r m o p h i l i s 5SrRNA has o n l y the c e n t r a l t e t r a m e r i c C-G-A-A sequence (66-67). Another i n t e r e s t i n g f e a -ture of B. s t e a r o t h e r m o p h i l i s ribosomal r e c o n s t i t u t i o n e x p e r i -ments i s that other b a c t e r i a l 5SrRNA molecules which possess a c e n t r a l C-G-A-A sequence can be i n c o r p o r a t e d i n the place of the n a t i v e type with r e s u l t i n g b i o l o g i c a l a c t i v i t y (68). These o b s e r v a t i o n s tend to imply that a s p e c i f i c sequence i s not en-t i r e l y c r i t i c a l f o r f u n c t i o n . - 30 -The T-^-C-G segment found i n almost a l l sequenced tRNA molecules can p o t e n t i a l l y form base p a i r s with the C-G-A-A region of 5SrRNA (65, 69). This has suggested that the b i n d -ing of tRNA to the ribosome during p r o t e i n s y n t h e s i s may i n -volve the base p a i r i n g of these two r e g i o n s . However, X-ray c r y s t a l l o g r a p h y (70-71), enzymatic a c c e s s i b i l i t y s t u d i e s (44), chemical modification»studies (44), and o l i g o n u c l e o t i d e b i n d -ing (72) a l l i n d i c a t e that the T-Y-C-G segment i s b u r i e d and t h e r e f o r e i n a c c e s s i b l e . The b i n d i n g of the C-G-A-A segment of 5SrRNA would r e q u i r e a c o n f o r m a t i o n a l change i n tRNA that exposes the T-y-C-G region (65). Numerous experiments have been designed to t e s t t h i s h y p o t h e s i s . S t u d i e s i n v o l v i n g tetramers of T-^-C-G or C-G-A-A tend to s u b s t a n t i a t e t h i s proposed i n -t e r a c t i o n between 5SrRNA and tRNA. The presence of the t e t r a -Phe mer T-y-C-G c o m p e t i t i v e l y i n h i b i t s the bi n d i n g of tRNA to E. c o l i ribosomes (73). The same tetramer a l s o binds s t r o n g l y to a 5SrRNA-ribosomal p r o t e i n complex (74). T h i s b i n d i n g i s reduced t e n - f o l d f o r f r e e SSrRMA i n d i c a t i n g t h at c o n f o r m a t i o n a l changes i n the molecule, due to ribosomal p r o t e i n s , f a c i l i t a t e b i n d i n g of the tetramer. A l t e r n a t i v e l y , the presence of t e t r a -mer i c C-G-A-A i n h i b i t s p o ly U d i r e c t e d p o l y p h e n y l a l a n i n e syn-t h e s i s of the ribosome' (75). Presumably i t i s competing with the C-G-A-A region of 5SrRNA f o r bi n d i n g to the T-^-C-G re g i o n of the incoming tRNA. There i s a l s o evidence that tRNA under-goes a c o n f o r m a t i o n a l change, which exposes i t s T-Y-C-G seg-ment, upon bi n d i n g to the mRNA codon (65). Equimolar amounts - 31 -o.f £-G-( 3H)A-( 3H)A w i l l bind to a 30S x phe - tRNA x EF-T u x GTP x polyU complex. With poly U absent o n l y about h as much tetramer binds. I t i s p o s s i b l e to r e c o n s t i t u t e f u n c t i o n a l 50S ribosomal subunits of E. c o l i where 5SrRNA i s added duri n g the l a s t step of the assembly (76). E l i m i n a t i o n of t h i s f i n a l step leaves an i n a c t i v e 47S ribosomal p a r t i c l e which l a c k s 5SrRNA and i s p a r t l y d e f i c i e n t i n four ribosomal p r o t e i n s known to a s s o c i a t e d i r e c t l y or i n d i r e c t l y with 5SrRNA. Experiments with these 47S p a r i t i c l e s have provided three important c l u e s to the f u n c t i o n -a l i t y of 5SrRNA. F i r s t , 5SrRNA i s e s s e n t i a l f o r poly U d i r e c t e d p o l y - p h e n y l a l a n i n e s y n t h e s i s . A 47S p a r t i c l e e x h i b i t s l e s s than 1% of the a c t i v i t y of a completely r e c o n s t i t u t e d 50S sub u n i t . Second, 5SrRNA i s l i k e l y to be i n p r o x i m i t y to the ribosomal A site.:.and P s i t e . Chloramphenicol, which binds to the A s i t e and C-A-C-C-A-acLeu fragments, s p e c i f i c f o r the P s i t e , does riot bind to 47S p a r t i c l e s . L a s t l y , 5SrRNA i s important f o r the Phe binding of Phe-tRNA to both the A s i t e and the P s i t e . No Phe EF-T enzymatic b i n d i n g of Phe-tRNA to the A s i t e was ob-served and IF dependent b i n d i n g of t h i s amino a c y l a t e d tRNA to the P s i t e was reduced by 66% f o r these 47S p a r t i c l e s . The i m p l i c a t i o n i s that there i s d i r e c t involvement between 5SrRNA Phe and the bi n d i n g of Phe-tRNA to the A s i t e . The r o l e of 5SrRNA with the P s i t e i s a p p a r e n t l y l e s s d i r e c t . The E. c o l i 5SrRNA molecule, at the ribosome, i s surrounded by ribosomal p r o t e i n s , s i n c e i t i s s t r o n g l y p r o t e c t e d from - 32 -degradation by r i b o n u c l e a s e and p a n c r e a t i c r i b o n u c l e a s e (77). Even a f t e r t r y p s i n d i g e s t i o n of the ribosomal p r o t e i n s the 5SrRNA remains s t r o n g l y p r o t e c t e d from r i b o n u c l e a s e d i g e s t i o n . This i n a c c e s s i b i l i t y to molecules the s i z e of degradation en-zymes suggests t h a t the t r a n s l a t i o n enzymes do not i n t e r a c t d i r e c t l y with 5SrRNA. Chemical m o d i f i c a t i o n with k e t h o x a l , a small molecule t h a t r e a c t s with s i n g l e stranded G r e s i d u e s i n d i c a t e s t h at G ^ of the c e n t r a l C-G-A-A segment i s not acces-s i b l e to e i t h e r ribosome bound or f r e e 5SrRNA (78). Only G ^ and G-j^ r e a c t r e a d i l y f o r both f r e e 5SrRNA and when i n c o r p o r a t e d i n t o the 50S ribosomal s u b u n i t . For 70S ribosomes G-^ i s par-t i a l l y p r o t e c t e d (79). Neither of these r e a c t i v e G r e s i d u e s appears to be f u n c t i o n a l l y s i g n i f i c a n t s i n c e 5SrRNA c h e m i c a l l y modified with kethoxal at G 4 1 and G^ 3 can be r e c o n s t i t u t e d i n t o 50S ribosomal subunits without e x t e n s i v e l o s s i n b i o l o g i c a l a c t i v i t y (78) . The strong p r o t e c t i o n of 5SrRNA, at the r i b o -some, from r i b o n u c l e a s e d i g e s t i o n and chemical m o d i f i c a t i o n by smaller molecules means t h a t the C-G-A-A segment, p o s t u l a t e d to i n t e r a c t with the T-f-C-G loop of tRNA, i s not a c c e s s i b l e . T h i s suggests that there may be some requirement f o r conforma-t i o n a l changes i n ribosomal p r o t e i n s and/or 5SrRNA i n order f o r the p o s t u l a t e d i n t e r a c t i o n to occur. E x t e n s i v e c o n f o r m a t i o n a l changes of the ribosomal p r o t e i n s occur d u r i n g m i l d d i s s o c i a -t i o n of the ribosome i n t o i t s two subunits through magnesium removal with e t h y l e n e d i a m i n e t e t r a a c e t a t e (EDTA) (80). T h i s i s ev i d e n t from changes i n the f a r u l t r a v i o l e t c i r c u l a r d i c h r o i s m - 33 -spectrum, an i n d i c a t i o n of a l t e r a t i o n s i n the h e l i c a l s t r u c t u r e of ribosomal p r o t e i n s due to subunit d i s s o c i a t i o n s . The near u l t r a v i o l e t c i r c u l a r d i c h r o i s m spectrum, due e x c l u s i v e l y to the three RNA molecules, i s unchanged by such d i s s o c i a t i o n . Only under extreme c o n d i t i o n s : o f ribosomal u n f o l d i n g was any con-f o r m a t i o n a l change i n the RNA observed by c i r c u l a r d i c h r o i s m . These experiments demonstrate the strong c o n f o r m a t i o n a l i n t e g -r i t y of the three RNA molecules i n the ribosome and p o s t u l a t e the p o s s i b i l i t y f o r e x t e n s i v e c o n f o r m a t i o n a l a l t e r a t i o n s i n ribosomal p r o t e i n s t r u c t u r e d u r i n g p r o t e i n s y n t h e s i s . Ribo-somal d i s s o c i a t i o n does not a l t e r kethoxal m o d i f i c a t i o n (81) nor does i t expose the C-G-A-A segment of 5SrRNA to enzymatic degradation (77). However, the p o s s i b i l i t y t h a t t r a n s l a t i o n enzymes or tRNA molecules can induce c o n f o r m a t i o n a l changes in surrounding ribosomal p r o t e i n s , which would expose the C-G-A-A region of 5SrRNA for b i n d i n g to the T-V-C-G segment of tRNA, cannot be r u l e d out. Treatment of E. c o l i ribosomes with high c o n c e n t r a t i o n s of l i t h i u m c h l o r i d e or ammonium c h l o r i d e r e l e a s e s 5SrRNA and some ribosomal p r o t e i n s (82-83). D i a l y s i s to reduce the s a l t con-c e n t r a t i o n r e s u l t s i n 7 0-80% reattachment of 5SrRNA to the ribosome core (84). Further experiments show that only three p r o t e i n s , L5; L18, and L25 are r e q u i r e d f o r reattachment. T h i s suggests that 5SrRNA and these three p r o t e i n s r e p r e s e n t a d i s -c r e t e u n i t of the ribosomal 50S s u b u n i t . The L5, L18, and L25 p r o t e i n s have a l s o been shown to have the s t r o n g e s t b i n d i n g - 34 -a f f i n i t y f o r f r e e 5SrRNA (85-86). Only two other 50S subunit p r o t e i n s , L20 and L3, bind very weakly to 5SrRNA (85). The a f f i n i t y of L18 f o r 5SrRNA i s the s t r o n g e s t . I t i s 16 times g r e a t e r than L25 and 100 times that of L5 (86). The f u n c t i o n s of L5 and L18 may be r e l a t e d s i n c e the a s s o c i a t i o n of L5 with 5SrRNA appears to be c o o p e r a t i v e l y s t i m u l a t e d by the presence of L18 while L25 does not e x h i b i t c o o p e r a t i v i t y with e i t h e r L5 or L18 (87) . Ribonuclease d i g e s t i o n i n v o l v i n g L18 and L25 bound 5SrRNA suggest t h a t the most important r e c o g n i t i o n region f o r both L18 and L25 i s the sequence 69-110 of 5SrRNA (88). Com-p e t i t i v e b i n d i n g s t u d i e s i n v o l v i n g ethidium bromide i n d i c a t e that these p r o t e i n s (L5, L18, and L25) are probably not b i n d -ing to the same s i t e (89). Ethidium bromide, which i n t e r c a l -ates between A-U p a i r s of double stranded regions of n u c l e i c a c i d s has 5 or 6 b i n d i n g s i t e s on 5SrRNA. An L18 x 5SrRNA com-plex causes a two-fold decrease in the number of bound ethidium bromides. A complex of L5 x 5SrRNA has a s l i g h t e f f e c t while L25 x 5SrRNA has no e f f e c t on ethidium bromide b i n d i n g . These o b s e r v a t i o n s suggest that L18 must be b l o c k i n g ethidium bromide bi n d i n g s i t e s at the same double stranded regions as ethidium bromide and t h a t the b i n d i n g s i t e s of the other two p r o t e i n s , L25 and L5, are i n d i f f e r e n t l o c a t i o n s . T h i s i m p l i e s t h a t the apparent c o o p e r a t i v i t y between L5 and L18 must be f a i r l y i n -d i r e c t . The e f f e c t s of ribosomal p r o t e i n s L5, L18, and L25 on the conformation of f r e e E. c o l i 5SrRNA have been i n v e s t i g a t e d - 35 -(90-92). The ribosomal p r o t e i n with the s t r o n g e s t a f f i n i t y f o r 5SrRNA, L18 , causes a marked in c r e a s e i n the u l t r a v i o l e t c i r -c u l a r d i c h r o i s m s p e c t r a l i n t e n s i t y . T h i s i s an i n d i c a t i o n of i n c r e a s e d secondary s t r u c t u r e f o r the RNA molecule. One study i n d i c a t e s t h a t an L25 x 5SrRNA complex g i v e s a s l i g h t , but s i g -n i f i c a n t , decrease suggesting a s m a l l r e d u c t i o n i n secondary s t r u c t u r e (91). The L5 x 5SrRNA complex has no e f f e c t on the near u l t r a v i o l e t c i r c u l a r d i c h r o i s m spectrum of 5SrRNA. A 5SrRNA complexed with a l l three p r o t e i n s g i v e s a spectrum which i s the sum of the above i n d i v i d u a l e f f e c t s . A c o n f o r m a t i o n a l change due to p r o t e i n b i n d i n g i s a l s o i n d i c a t e d from the enzym-a t i c and chemical a c c e s s i b i l i t y to the U 8 7 - C 8 8 ~ U 8 9 r e < 3 i ° n °f 5SrRNA (88). In an L18 x L25 x 5SrRNA complex t h i s r e g i o n i s a c c e s s i b l e to both p a n c r e a t i c r i b o n u c l e a s e and c a r b o d i i m i d e . Free 5SrRNA i s not a c c e s s i b l e to e i t h e r of these agents i n t h i s r e g i o n . The changes i n molecular conformation due to the b i n d -ing of ribosomal p r o t e i n s suggest that f r e e 5SrRNA i n aqueous s o l u t i o n has a d i f f e r e n t conformation than when i t i s p a r t of the ribosome. A complex of L5, L18 , and L25 p l u s 5SrRNA e x h i b i t s in. v i t r o GTPase a c t i v i t y (92). Cross l i n k i n g experiments using 4-mer-captobutyrimide i n d i c a t e formation of d i s u l f i d e bonded c r o s s l i n k s between L5-L7 and L5-L12 (93). P r o t e i n s L7 and L12 are known to be l o c a t e d near the p e p t i d y l t r a n s f e r a s e center (L16, L l l , and L2) (94). T h i s suggests a r e l a t i o n s h i p between the - 36 -p e p t i d y l t r a n s f e r a s e and GTPase c e n t e r s of the 50S ribosomal s u b u n i t . I t a l s o i m p l i e s that 5SrRNA i s v i c i n a l to these two c a t a l y t i c s i t e s . A s t a b l e complex between E. c o l i 23SrRNA and 5SrRNA can be formed i n the presence of the ribosomal p r o t e i n s L18, L25 , L6, and L2 (95). Only 8 50S ribosomal p r o t e i n s independently bind to 23SrRNA ( i n c l u d i n g L2 and L 6 ) . L18 and L25, which bind s t r o n g l y to 5SrRNA, have no a f f i n i t y f o r 23SrRNA. T h i s i n d i -c a t e s a c o o p e r a t i v e i n t e r a c t i o n between 5SrRNA, 23SrRNA and the four ribosomal p r o t e i n s . When the 50S subunit i s s p l i t with high s a l t c o n c e n t r a t i o n under c o n d i t i o n s of c o n t r o l l e d pancre-a t i c r i b o n u c l e a s e d i g e s t i o n fragments of 23SrRNA r e s u l t ; an 18S (3' end) and a 13S fragment (96). An 18SrRNA:5SrRNA complex can be formed with the above mentioned p r o t e i n s i n d i c a t i n g t h a t the o v e r a l l i n t e g r i t y of the 23SrRNA i s not r e q u i r e d f o r a s s o c i -a t i o n with 5SrRNA. No a s s o c i a t i o n of 5SrRNA with e i t h e r the 13S fragment of 23SrRNA or with the 16SrRNA (from the 30S sub-un i t ) has been observed. A proposed s i t e of i n t e r a c t i o n be-tween 5SrRNA and 23SrRNA i s the 72-83 sequence of 5SrRNA which i s h i g h l y conserved i n p r o c a r y o t e s . A fragment of 23SrRNA, a c c e s s i b l e to kethoxal m o d i f i c a t i o n , i s complementary to t h i s 5SrRNA segment (96) . The above remarks r e p r e s e n t c u r r e n t understanding of 5SrRNA f u n c t i o n at the ribosome. Based on sequence homology s t u d i e s and o l i g o n u c l e o t i d e experiments a l i k e l y f u n c t i o n a l r o l e f o r 5SrRNA i s shown i n Fig u r e 1.14; namely, the r e c o g n i t i o n of the - 37 -u a o 3'—U-U-U—5" F i g u r e 1.14. A proposed model f o r the f u n c t i o n of 5SrRNA. The model r e q u i r e s t h a t there be an induced c o n f o r m a t i o n a l change of the G-C-y-T region of tRNA which allows i t to hydrogen bond to the C-G-A-A segment of 5SrRNA (69). T-y-C-G region of tRNA duri n g i t s bi n d i n g to the ribosomal A s i t e . 5SrRNA and three ribosomal p r o t e i n s (L5, L18, and L25) apparently form a d i s c r e t e u n i t w i t h i n the ribosome s t r o n g l y p r o t e c t i n g i t from enzymatic d e g r a d a t i o n . Besides protein-RNA i n t e r a c t i o n s there i s a l s o evidence f o r an RNA-RNA i n t e r a c t i o n between 5SrRNA and 23SrRNA. M u l t i p l e f u n c t i o n s f o r E. c o l i 5SrRNA cannot be r u l e d out as e v i d e n t from the apparent prox-i m i t y of 5SrRNA to the p e p t i d y l t r a n s f e r a s e and GTPase c a t a l y t i c c e n t e r s of the 50S s u b u n i t . The work which w i l l be presented i n t h i s t h e s i s i s con-cerned e x c l u s i v e l y with the use of 1 9F-nmr and l a s e r Raman spectroscopy to i n t e r p r e t c o n f o r m a t i o n a l p r o p e r t i e s of f r e e E. c o l i 5SrRNA i n aqueous s o l u t i o n . Much i n f o r m a t i o n about the macromolecular conformation of f r e e 5SrRNA i s a v a i l a b l e i n the - 38 -l i t e r a t u r e . I t has a s e d i m e n t a t i o n c o e f f i c i e n t (S2Q w ) o f a p p r o x i m a t e l y 4i.5S-4.8S which i s f a i r l y i n s e n s i t i v e t o c o u n t e r -i o n s ( 9 7 ) . T h i s i s i n d i c a t i v e o f a f a i r l y r i g i d s t r u c t u r e . A c c o r d i n g t o the most r e c e n t s m a l l a n g l e X-ray s c a t t e r i n g s t u d -i e s 5SrRNA p r o b a b l y assumes the shape of a p r o l a t e e l i p s o i d w i t h an a x i a l r a t i o o f 5:1 i n s o l u t i o n (98). I t s r a d i u s o f g y r a t i o n (R g) has been e s t i m a t e d t o be 34±1.5 angstroms. T h i s compares w i t h a t h e o r e t i c a l R v a l u e o f 48 angstroms f o r a y s t r a i g h t r i g i d double h e l i x o f 120 bases. 5SrRNA i n s o l u t i o n a l s o appears t o be more asymmetric than tRNA and p r o b a b l y b u l g e s a t one end (98-99). Free 5SrRNA i s known t o e x i s t i n two s t a b l e forms under d e n a t u r i n g c o n d i t i o n s (7 molar Urea and 0.01 molar EDTA) (100-101). Both forms are s t a b l e and can be s e p a r a t e d by Sephadex G-100 or m e t h y l a t e d bovine serum a l b u m i n -c o a t e d k i e s e l g u h r (MAK) chromatography. The B form w i l l not r e i n c o r p o r a t e d u r i n g r e c o n s t i t u t i o n o f 50S r i b o s o m a l s u b u n i t s w h i l e the A form behaves l i k e n a t i v e 5SrRNA. Under r e n a t u r i n g c o n d i t i o n s , i n the presence o f magnesium, the B form can be c o n v e r t e d t o the A form. Such m u l t i p l e c o n f o r m a t i o n s f o r RNA m o l e c u l e s are not uncommon. Numerous examples of b i o l o g i c a l l y i n a c t i v e forms of tRNA have been p r e s e n t e d i n the l i t e r a t u r e (102) . U l t r a v i o l e t s p e c t r o s c o p y and 'H-nmt s p e c t r o s c o p y have been used t o o b t a i n g e n e r a l i z e d base p a i r i n g p r o p e r t i e s o f E. c o l i 5SrRNA. O p t i c a l m e l t i n g c u r v e s a t 260 m i l l i m i c r o n s i n -d i c a t e t h a t 5SrRNA has a r e l a t i v e l y h i g h m e l t i n g temperature - 3 9 -compared to tRNA and the presence of magnesium and monovalent c a t i o n s i n c r e a s e s the melting temperature by about 10 degrees c e n t i g r a d e (103). I t i s a l s o p o s s i b l e to estimate the t o t a l number of base p a i r s , the number which are G-C p a i r s , and the number of A-U p a i r s under a v a r i e t y of b u f f e r c o n d i t i o n s . T h i s data i s compiled i n Table 1.2. The l a r g e number of G-C base p a i r s (60-68% of the t o t a l ) e x p l a i n s the high m e l t i n g temper-ature of E. c o l i 5SrRNA. The 'H-nmr s p e c t r a a l s o i n d i c a t e e x t e n s i v e G-C base p a i r i n g (24 G-C p a i r s ) (108). However, the low number of p r e d i c t e d A-U p a i r s (only 4) and the i n a b i l i t y to d e t e c t s t r u c t u r a l a l t e r a t i o n s between samples c o n t a i n i n g magnesium and those without i s i n c o n s i s t e n t with the a v a i l a b l e o p t i c a l . d a t a . Chemical m o d i f i c a t i o n s t u d i e s have been used to probe the secondary s t r u c t u r e of E. c o l i 5SrRNA. The most widely used chemical m o d i f i e r s of RNA molecules are k e t h o x a l , g l y o x a l , n i t r o u s a c i d , monoperphthalic a c i d and methoxyamine. Both keth-o x a l and g l y o x a l r e a c t with unpaired guanines. The most r e -a c t i v e s i t e i n n a t i v e 5SrRNA i s G ^ while the second most r e a c t i v e s i t e i s G ^ (109). A f t e r 45 minutes treatment of 5SrRNA with kethoxal 100% of G 4 1 has reacted while only about 40% of G^-j i s m o d i f i e d . Exposures of t h i s time d u r a t i o n w i l l reduce ribosomal a f f i n i t y . T h i s i s probably due to chemical m o d i f i c a t i o n of G ^ s i n c e f o r m y l a t i o n of o n l y G ^ , with g l y -o x a l , does not g r e a t l y a f f e c t ribosomal a f f i n i t y (more than 70% r e t e n t i o n ) (110). In denatured E. c o l i 5SrRNA (B form) NaCl(M)\ MgCl.,(M) A-U G-C T o t a l B.P. —m- (°C) Melt. Range (°C) Reference 0.1 None . 13 28 41 — — 104 0 .15 None 38-39 60 (est.) 40-80 (est.) 105 2.0 None 11-14 22-25 36 70 (est. ) 60-90 (est.) 106 None 0.01 18 27 45 80 (est.) 65-90 (est.) 107 Denatured or B Form None 0.01 17 26 43 80 (est.) 65-90 (est.) 107 0.15 None 31 60 (est.) 40-80 (est.) 105 Accurate T d e t e r m i n a t i o n from d e r i v a t i v e curve m 1) low s a l t (0.01M KC1, 0.001M T r i s - H C l , pH7) 103 T = 5 6 0 C m 2) p h y s i o l o g i c a l s a l t (0.13M KC1, 0.09M NaCl, 103 0.06M MgCl 2, 0.001M T r i s - H C l , pH7) T =66°C m Table 1.2. Shown above are estimates of the base p a i r i n g i n E. c o l i 5SrRNA obtained by o p t i c a l s p e c t r o s c o p y . As i n d i c a t e d they are at v a r i o u s c o n c e n t r a t i o n s of NaCl and MgCl 2 f o r both the n a t i v e and denatured forms. The accurate melting temperature, T , at physi-o l o g i c a l s a l t c o n c e n t r a t i o n s and at low s a l t c o n c e n t r a t i o n s i s a l s o shown. The most s i g n i f i c a n t f e a t u r e s of t h i s t a b l e are the extensive G-C base p a i r i n g and the apparent in c r e a s e i n T m due to the presence of monovalent and d i v a l e n t c a t i o n s . - 41 -i s n o t a c c e s s i b l e t o t h e s e two c h e m i c a l m o d i f i e r s w h i l e G g l becomes e x p o s e d ( 1 1 0 ) . I t has been s u g g e s t e d t h a t d u r i n g t h e r e n a t u r a t i o n - d e n a t u r a t i o n p r o c e s s G ^ and G ^ p l a y an i m -p o r t a n t r o l e . N i t r o u s a c i d i s a l e s s s p e c i f i c c h e m i c a l m o d i -f i e r . I t d e a m i n a t e s e x p o s e d a d e n i n e s , g u a n i n e s , and c y t o s i n e s . The most r e a c t i v e r e g i o n s o f 5SrRNA a r e b a s e s 34-41 and 44-55 ( 1 0 9 ) . T hese m o d i f i c a t i o n s r e s u l t i n l o s s o f a f f i n i t y f o r r i b o -s o m a l p r o t e i n s b u t t h e l a c k o f s p e c i f i c i t y makes i n t e r p r e t a t i o n d i f f i c u l t . M e t h o x y a m i n e i s s p e c i f i c f o r a c c e s s i b l e c y t o s i n e s i n RNA. The most e x p o s e d r e g i o n i n E. c o l i 5SrRNA i s a r o u n d p o s i -t i o n s 35-39 ( 1 0 9 ) . The r e a c t i o n o f E. c o l i 5SrRNA w i t h m e t h o x y -amine c a u s e s r a p i d l o s s o f a f f i n i t y f o r r i b o s o m a l p r o t e i n s , b u t a g a i n i n t e r p r e t a t i o n i s d i f f i c u l t due t o t h e l a r g e number o f r e a c t i v e s i t e s . O x i d a t i o n w i t h m o n o p e r p h t h a l i c a c i d i n d i c a t e s t h a t 10 o f t h e 23 a d e n i n e s o f 5SrRNA a r e u n p a i r e d ( 1 1 1 ) . T h i s a l s o c a u s e s a r a p i d l o s s i n r i b o s o m a l a f f i n i t y . I t h a s been p o s t u l a t e d t h a t two o f t h e a d e n i n e s w h i c h r e a c t a r e t h o s e a s s o -c i a t e d w i t h t h e G-C-A-A segm e n t . The b a s i s f o r t h i s a r g u m e n t i s t h e i n a b i l i t y t o b i n d T-y-C-G t o 5SrRNA a f t e r o x i d a t i o n o f t h e a d e n i n e s ( 1 1 1 ) . A n o t h e r more r e c e n t l y d e v e l o p e d c h e m i c a l m o d i -f i e r i s 1 , 4 - p h e n y l d i g l y o x a l (PDG). U n l i k e k e t h o x a l and o t h e r a l i p h a t i c d i c a r b o n y l s w h i c h o n l y r e a c t w i t h u n s t a c k e d g u a n i n e s , PDG c a n r e a c t w i t h b a s e p a i r e d g u a n i n e s ( 1 1 2 ) . I t s a r o m a t i c c h a r a c t e r a l l o w s i t t o i n t e r c a l a t e i n t o d o u b l e h e l i c a l RNA r e -g i o n s p r o d u c i n g c r o s s l i n k a g e s b e t w e e n n e i g h b o r i n g g u a n i n e s . - 42 -T h i s m o d i f i e r g i v e s evidence f o r a h e l i c a l stem formed by the two ends of E. c o l i 5SrRNA by showing that G 2 and G-^ 2 become cr o s s l i n k e d with PDG. T h i s i s only p o s s i b l e i f these two bases are i n the d i r e c t neighborhood across the l a r g e groove of the double h e l i x and are about 5 base p a i r s a p a r t . I t does however r e q u i r e that the two bases unstack and protrude s l i g h t l y from the h e l i x upon li n k a g e with PDG. The major s i t e s of chemical r e a c t i v i t y i n E. c o l i 5SrRNA mentioned above are summarized i n F i g u r e 1.15. C l e a r l y the most r e a c t i v e regions are segments 34-41 and 44-51 which suggest s i n g l e stranded loops i n these r e g i o n s . V a r i o u s r i b o n u c l e a s e enzymes are a l s o s e n s i t i v e to RNA secondary s t r u c t u r e . Sheep kidney r i b o n u c l e a s e , which has no p r e f e r e n t i a l s p e c i f i c i t y f o r p u r i n e s or p y r i m i d i n e s , w i l l not hydrolyze double stranded h e l i c e s and i s even reta r d e d by stacked s i n g l e stranded loops (113). T h i s enzyme i s p a r t i c u l a r l y r e a c -t i v e i n the v i c i n i t y of G ^ (113). The G ^ residue i s a l s o most a c c e s s i b l e f o r r i b o n u c l e a s e IV and r i b o n u c l e a s e T-^  (114-116). These and other major enzymatic cleavage s i t e s (117) are sum-marized in F i g u r e 1.15. Another method, which has i d e n t i f i e d s i n g l e stranded regions about 39-50 i n E. c o l i 5SrRNA, c o n s i s -tent with chemical m o d i f i c a t i o n and r i b o n u c l e a s e s t u d i e s , i s the b i n d i n g of complementary oligomers (118). T h i s i s a l s o shown in F i g u r e 1.15. - 43 -1. U 2. G 3. C 4. C 5. U 6. G 7. 8. G C 9. G 10. G 11. C 12. C "m" G "k" and "g" »-14. U 15. A ~ 16. G * 1 7 . C .18 . G "~*'19. C 20. G 21. G 22. U 23. G 24. G 25. U 26. C 27. c 28. c 29. A 30. C 31. C 32. U 33. G • 3 4 - A ' m" ' 35. C ' n" and "m" 36. C ' n" and "m" 37. c • n" and "m" 38. C ' n" and "m" 39. A ' n" 40. U 1 i 4 1 • G "k * 42. C "m 43. C "m > 4 4 • G "n * 45. A "n 46. A "n I 4 7 • C "n * 48. U 49. c "n „ 50. A "n * 51. G "n 52. A 53. A 54. G 55. U t 5 6- G * 57. A 58. A 59. A 60. C 61. G "9 62. C 63. C 64. G 65. U 66. A 67. G 68. C G * 70. C 71. C 72. G 73. A 74. U 75. G 76. G 77. U 78. A 79. G 80. U " g " , and "n" " and "m" and "m" (denatured only)  81. 82. 83. 84. 85. 86. " 87. 88. 89. 90. 91. 92. 93. 94. 95. 96. 97. 98. 99. 100. 101. 102. 103. 104. 105. 106. 107. 108. 109. 110. 111. 112. 113. 114. 115. 116. 117. 118. 119. 120. J F i g u r e 1.15. A summary of the regions of 5SrRNA which are most a c c e s s i b l e to enzymes and chemical modifying agents. The chemical m o d i f i e r s considered are kethoxal ("k"), g l y o x a l ("g"), n i t r o u s a c i d ("n"), and methoxyamine ("m"). The arrows i n d i c a t e the s i t e s of enzymatic cleavage. The enzyme respon-s i b l e f o r cleavage i n each s p e c i f i c r e g ion of 5SrRNA i s not i n d i c a t e d (see t e x t ) . The bracketed regions i n d i c a t e s i t e s of o l i g o n u c l e o t i d e b i n d i n g . - 44 -Unlike tRNA no unique secondary structure for E. c o l i 5SrRNA can be ascertained. The d i f f i c u l t y with i n f e r r i n g a secondary structure for this molecule i s mostly due to three factors. F i r s t , 5SrRNA i s about half again larger than tRNA. Computer studies have shown that the number of possible struc-tures increase exponentially with size of the molecule (117). Secondly, the function of 5SrRNA is s t i l l not well understood. Any proposed secondary structure i s therefore debatable on grounds of functional uncertainty. Lastly, i t i s not clear that free 5SrRNA has the same conformation as in i t s native ribosomal environment. As previously mentioned u l t r a v i o l e t c i r c u l a r d i -chroism studies involving the d i s s o c i a t i o n of intact E. c o l i ribosomes into 30S and 50S subunits indicate conformational i n -teg r i t y of the three RNA molecules i s retained (80). However, binding studies involving free 5SrRNA and the ribosomal protein L18, shows changes in the spectrum indicative of substantial alterations in conformation (90-92). A number of secondary structures for E. c o l i 5SrRNA have been proposed but none are e n t i r e l y consistent with a l l the available physical, chemical, and biochemical data (119-122, 108). Two models which are most consistent with the experimen-t a l results are shown in Figure 1.16. The formulation of the f i r s t model (Figure 1.16A) involved a comparative study of se-quences from known procaryotic 5SrRNA molecules (120). It assumes that functionally equivalent 5SrRNA molecules from d i f -ferent procarotes w i l l have similar structures. Based upon this - 45 -A B u pU - A G-C C-G C-G fT \ / \ .e-c*cx G-c p y S c c u 6 f l c « ^ Voeue- 8 \ g i g m i M e * « A G c « y 5 o e c c e c OACU • 5-c A - A - O - C C - G C-G (G)U G U-A //HO*'-'1''-" A c - « _ A - U GG CgC-G »L> UGGU-A " *-AGyC CAC 1 , G C G U A C C C C " C G A * C * A A A C G - U A G U G U G G G G " * C-G C-G G-U G C Figure 1.16. Two proposed models of the secondary s t r u c t u r e of E. c o l i 5SrRNA which best f i t the experimental data. The model proposed by Fox and Woese (1.16A) (120) has only 25 base p a i r s which i s a low value compared to the o p t i c a l data. The model proposed by Luoma and M a r s h a l l (1.16B) i s remarkably s i m i l a r to the c l o v e r l e a f ' s e c o n d a r y s t r u c t u r e of tRNA (121). study the model p o s t u l a t e s the e x i s t e n c e of four conserved h e l i -c a l r e g i o n s ; a molecular s t a l k (1-10 base p a i r e d to 119-110), a tuned h e l i x (18-23 base p a i r e d to 65-60), a common arm (31-34 - 46 -base p a i r e d to 51-48) and a p r o c a r y o t i c loop (82-86 base p a i r e d to 94-90). T h i s s t r u c t u r e i s c o n s i s t e n t with most r i b o n u c l e a s e d i g e s t i o n and chemical m o d i f i c a t i o n experiments provided that allowances are made f o r the p r o t e c t i o n of c e r t a i n r e s i d u e s due to t e r t i a r y i n t e r a c t i o n s . The model f o r E. c o l i has a t o t a l of 25 base p a i r s (5 A-U, 17 G-C, and 3 G-U). This i s i n c o n s i s t e n t with o p t i c a l data (see Table 1.2) which suggest more ex t e n s i v e base p a i r i n g . An i n t e r p r e t a t i o n of A and B co n f o r m a t i o n a l changes f o r 5SrRNA, based on t h i s model, have r e c e n t l y been proposed (123). I t p o s t u l a t e s a d i s r u p t i o n of the h e l i c a l stem region of the p r o c a r y o t i c loop to form a new h e l i c a l segment i n v o l v i n g bases 88-82 p a i r e d to bases 41-47. T h i s n e c e s s i t a t e s d i s r u p t i o n of the tuned h e l i x and the common arm (see Figure 1.16A) . More r e c e n t l y , G. Luoma and A. M a r s h a l l of t h i s l a b o r a t o r y have proposed a u n i v e r s a l secondary s t r u c t u r e f o r RNA molecules of t h i s l e n g t h (121) . I t i s remarkably s i m i l a r to the c l o v e r l e a f of tRNA as shown i n Figu r e 1.16B. The model has a t o t a l of 37 base p a i r s (9 A-U, 22 G-C, and 6 G-U) which i s c o n s i s t e n t with the o p t i c a l d ata. Ribonuclease a c c e s s i b i l i t y and chemical m o d i f i -c a t i o n experiments are a l s o c o n s i s t e n t with s i n g l e stranded or s t r a i n e d regions of t h i s s t r u c t u r e . Besides the work presented i n t h i s t h e s i s two other nmr s t u d i e s and one l a s e r Raman study of 5SrRNA have appeared i n the l i t e r a t u r e . One study i n v o l v e d 'H-nmr spectroscopy of the low f i e l d exchangeable r i n g NH protons of E. c o l i 5SrRNA (108). - 47 -Spectra at v a r i o u s temperatures are shown i n Figure.1.17. They have determined that there are 4 A-U and 24 G-C base p a i r s . However, these s p e c t r a are p o o r l y r e s o l v e d and as p r e v i o u s l y mentioned there i s no s i g n i f i c a n t d i f f e r e n c e between the mag-nesium c o n t a i n i n g sample and the one not c o n t a i n i n g magnesium. A low value f o r the degree of base p a i r i n g , as compared to the o p t i c a l data, may be a t t r i b u t e d to the i n t e g r a t i o n technique Phe which i s known to give low values f o r tRNA (124). The onl y other p u b l i s h e d nmr study used 1 3C-nmr of e n r i c h e d C-4 l a b e l e d u r a c i l . The in v i v o i n c o r p o r a t i o n i n t o Salmonella typhimurium 5SrRNA, whose sequence i s s i m i l a r to E. c o l i 5SrRNA, provided the s p e c t r a shown i n Fig u r e 1.18 (125). The 37°C spectrum shows 8 w e l l r e s o l v e d peaks and i n d i c a t e s that at l e a s t 75% of the u r a c i l s are i n v o l v e d i n secondary i n t e r a c t i o n s . Most r e c e n t l y a l a s e r - Raman spectrum of E. c o l i 5SrRNA, i n aqueous s o l u t i o n , has appeared i n the l i t e r a t u r e . I t i s shown i n Fig u r e 1.19 (126). T h i s spectrum has been compared with a number of d i f -f e r e n t tRNA molecules i n order to i n f e r some s p e c i f i c conforma-t i o n a l i n f o r m a t i o n about 5SrRNA. T h e i r r e s u l t s i n d i c a t e more Phe e f f e c t i v e s t a c k i n g of G r e s i d u e s than i n tRNA and the f r a c -t i o n of G re s i d u e s i n the stem must be l a r g e r than expected. On the other hand the f r a c t i o n of stacked A r e s i d u e s must be Phe l e s s i n 5SrRNA than i n tRNA . - 48 -RRMXDSS) F i g u r e 1.17. 300 megahertz low f i e l d 'H-nmr s p e c t r a of E. c o l i 5SrRNA at v a r i o u s temperatures. The sample c o n c e n t r a t i o n was 1.4 m i l l i m o l a r . The aqueous b u f f e r contained 0.01 molar caco-d y l a t e (pH 7) and 0.1 molar NaCl. The presence of magnesium had no e f f e c t on the s p e c t r a (108). - 49 -6 4 I Figure 1.18. The 1 3C-nmr spectrum of the C-4 u r i d i n e carbons of Salmonella typhimurium 5SrRNA at 37°C (A) and 75°C (B). The : sample was m i l l i m o l a r . An aqueous b u f f e r of 5 m i l l i m o l a r d i -t h i o t h r e i t o l (pH 7.4), c o n t a i n i n g 40 m i l l i m o l a r magnesium c h l o r -ide and 2 m i l l i m o l a r EDTA, was used. Spectrum A r e q u i r e d 18,000 t r a n s i e n t s while spectrum B i s from 14,000 t r a n s i e n t s (125). There were three major o b j e c t i v e s i n w r i t i n g t h i s chapter: f i r s t , to convey to the reader the reasons f o r for m u l a t i n g t h i s study; second, to provide the reader with background informa-t i o n about the s t r u c t u r e and f u n c t i o n of E. c o l i 5SrRNA which i s a v a i l a b l e i n the l i t e r a t u r e ; l a s t l y , to acquaint the reader with p h y s i c a l and chemical techniques that have been a p p l i e d to the e l u c i d a t i o n of the s t r u c t u r e and f u n c t i o n of E. c o l i 5SrRNA. The remainder of t h i s t h e s i s w i l l c o n s i d e r the u t i l i t y of - 50 -1800 1600 1400 1200 1000 800 600 400 C M " 1 Figure 1.19. A l a s e r Raman spectrum of a 5% aqueous s o l u t i o n of E. c o l i 5SrRNA (126). l 9F-nmr spectroscopy and l a s e r Raman spectroscopy f o r i n t e r -p r e t i n g conformation p r o p e r t i e s of E. c o l i 5SrRNA. - 51 -REFERENCES: CHAPTER 1 1. R.A. 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The f i r s t c o n s i d e r a t i o n was the growth of E. c o l i , i n the presence or absence of 5-FU, on c h e m i c a l l y d e f i n e d minimal media ( s e c t i o n 2.2). Next the b a c t e r i a l 5SrRNA (or FU-5SrRNA) was i s o l a t e d by e x t r a c t i o n and chromatographic procedures. The i n i t i a l f e a s i b i l i t y of t h i s study was d e t e r -mined by two s p e c i f i c experiments. The f i r s t one i n v o l v e d de-te r m i n a t i o n of the amount of r a d i o a c t i v e 5-FU ( l a b e l e d with carbon-14 at the 2-carbon p o s i t i o n of 5-FU) i n c o r p o r a t e d i n t o the b a c t e r i a l tRNA ( s e c t i o n 2.6). T h i s experiment showed that the i n c o r p o r a t i o n was e x t e n s i v e . The second f e a s i b i l i t y exper-iment concerned the obtainment of an 1 9F-nmr spectrum of h e t e r -ogeneous tRNA from E. c o l i . A f t e r p r a c t i c a b i l i t y was e s t a b -l i s h e d a s p e c i f i c RNA molecule was s e l e c t e d f o r f u r t h e r study. The choice of 5SrRNA was determined l a r g e l y by the r e l a t i v e l y l a r g e sample requirement f o r 1 9F-nmr spectroscopy. For an ad-equate s i g n a l to noise r a t i o i n a convenient amount of time on the departmental V a r i a n XL-100 spectrometer, 90 m i l l i g r a m s of sample i n 3 m i l l i l i t e r s of b u f f e r was r e q u i r e d . At a l a t e r stage of t h i s study a Bruker HX-270 spectrometer ( U n i v e r s i t y - 59 -of A l b e r t a b i o c h e m i s t r y department) was employed. Then on l y 15 m i l l i g r a m s of 5SrRNA i n 0.5 m i l l i l i t e r s of b u f f e r was r e q u i r e d . The 1 9F-nmr s p e c t r a are presented i n s e c t i o n 2.7. The concern about p o s s i b l e a l t e r a t i o n s i n 5SrRNA conforma-t i o n due to 5-FU i n c o r p o r a t i o n l e d to the second technique em-ployed i n t h i s t h e s i s , l a s e r Raman spectroscopy. The Raman spectrum of a s p e c i f i c RNA i s the sum of the v i b r a t i o n a l con-t r i b u t i o n s from a l l the bases plu s v i b r a t i o n a l components from the ribophosphate backbone. Conformational change a f f e c t s the i n t e n s i t i e s of many of these v i b r a t i o n a l components. Raman sp e c t r a of n a t i v e 5SrRNA and FU-5SrRNA are given i n s e c t i o n 2.8. A l s o c o n s i d e r e d i n the f i n a l s e c t i o n are the e f f e c t s of 5 - f l u o r o - s u b s t i t u t i o n on the Raman spectrum of the u r a c i l and u r i d i n e bases. - 60 -2.2 B a c t e r i a l Growth C u l t u r e s of E. c o l i B c e l l s , provided by the U.B.C. micro-b i o l o g y department, were grown on a c h e m i c a l l y d e f i n e d minimal media (1). The c o n s t i t u e n t s f o r one l i t e r of media are given in Table 2.1. These chemicals were weighed, d i s s o l v e d i n the Chemical Amount (grams) Potassium Phosphate D i b a s i c 7 Potassium Phosphate Monobasic 3 Magnesium S u l f a t e 0.1 Ammonium S u l f a t e 1 Sodium C i t r a t e 0.5 Glucose 2 Table 2.1. The chemical composition of one l i t e r of minimal media (1). ap p r o p r i a t e volume of d e i o n i z e d water, mixed, and au t o c l a v e d . T h i s l i q u i d media was used f o r a l l b a c t e r i a grown i n t h i s l a b -o r a t o r y . S o l i d media f o r s l a n t s and p e t r i d i s h e s contained 0.2% agar. A requirement f o r r e l a t i v e l y l a r g e q u a n t i t i e s of 5SrRNA made necessary the use of fermentation d e v i c e s of v a r i o u s capa-c i t i e s . The fermentors employed dur i n g v a r i o u s stages-of t h i s work are l i s t e d i n Table 2.2. The y i e l d for c e l l s t r e a t e d with 5-FU was about one gram (wet weight) per l i t e r of media while untreated c e l l s gave y i e l d s of at l e a s t two grams per l i t e r . S u i t a b l e i n o c u l a were u s u a l l y prepared i n erlenmeyer f l a s k s the n i g h t before a fermentation. They were allowed to grow ove r n i g h t at room temperature. The s i z e of the inoculum - 61 -Fermentation Device C a p a c i t y ( l i t e r s ) Erylenmeyer Flask 1-4 New Brunswick Batch Fermentor (Chemistry Department, U n i v e r s i t y of B r i t i s h Columbia) 10 New Brunswick Batch Fermentor (Microbiology Department, U n i v e r s i t y of B r i t i s h Columbia) 25 New Brunswick Batch Fermentor (Food Sci e n c e s , U n i v e r s i t y of B r i t i s h Columbia) 60 New Brunswick Batch and Continuous Flow 100 Fermentor (Biochemistry Department, U n i v e r s i t y of Washington, S e a t t l e , Washington) Table 2.2. The fermentation d e v i c e s used d u r i n g v a r i o u s stages of t h i s work. represented at l e a s t 3% of the fermentor volume. Larger i n o c u l a were d e s i r a b l e s i n c e they markedly diminished the i n i t i a l l a g phase p r i o r to e x p o n e n t i a l growth and reduced the p o s s i b i l i t y of contamination. In a l l cases the fermentation temperature was 37 degrees c e n t i g r a d e and growth was with constant a g i t a -t i o n and a e r a t i o n . Progress of b a c t e r i a l growth was monitored by measurement of the media's d i s p e r s i o n at 686 m i l l i m i c r o n s ( A 6 8 6 ) u s u a l l y on a G i l f o r d 240 spectrometer. T h i s i n v o l v e d withdrawing two m i l l i l i t e r a l i q u o t s at v a r i o u s time i n t e r v a l s and determining the media's t u r b i d i t y from i t s &§QQ v a l u e . The growth curves f o r untreated and 5-FU t r e a t e d E. c o l i B c e l l s are shown in F i g u r e 2.1. Untreated b a c t e r i a grew to an A, R f-- 62 -Time ( h o u r s ) F i g u r e 2.1. B a c t e r i a l growth curves of E. c o l i B grown on minimal media. F i g u r e 2.IA shows normal e x p o n e n t i a l growth. In F i g u r e 2.IB 25 m i l l i g r a m s of 5-FU was added to each l i t e r of media duri n g e a r l y e x p o n e n t i a l growth (Agg^ = 0.3). The reduced lagrphase, p r i o r to e x p o n e n t i a l growth, f o r Fig u r e 2.IA i s due to the a d d i t i o n of a l a r g e r inoculum. - 63 -of about 0.85 before l e v e l i n g to a s t a t i o n a r y phase. The a d d i -t i o n o f 5-FU (25 m i l l i g r a m s per l i t e r o f media) d u r i n g e a r l y ex-p o n e n t i a l growth ( Aggg - 0.3) caused i n h i b i t i o n of c e l l growth and apparent c e l l d e s t r u c t i o n . Media t u r b i d i t y would continue i n c r e a s i n g u n t i l about one hour a f t e r the a d d i t i o n of 5-FU. This was followed by a 50% r e d u c t i o n i n t u r b i d i t y p r i o r to a s t a t i o n a r y phase. T h i s i n d i c a t e d c e l l u l a r d e s t r u c t i o n due to the presence of 5-FU. In support of t h i s hypothesis was the ap-parent i n c r e a s e in v i s c o s i t y of the media which l e d to e x t e n s i v e foaming s h o r t l y a f t e r the a d d i t i o n of 5-FU. No foaming was ob-served i n b a c t e r i a grown without 5-FU. The foaming was presum-ably caused by the r e l e a s e of h i g h l y v i s c o u s d e o x y r i b o n u c l e i c a c i d and p r o t e i n m a t e r i a l due to c e l l l y s i s . A f t e r a d d i t i o n of 5-FU a g i t a t i o n and a e r a t i o n was continued f o r three hours. The c e l l s were u s u a l l y harvested with a Sharpies continuous flow c e n t r i f u g e . The harvested c e l l s were washed two times with 0.01 molar t r i s * H C l (pH 7.4) c o n t a i n i n g 0.01 molar magnesium acetate and s t o r e d at -20 degrees c e n t i g r a d e . The r e d u c t i o n i n the b a c t e r i a l p o p u l a t i o n due to the pre -sence of 5-FU caused concern about the extent of i n c o r p o r a t i o n i n t o b a c t e r i a l RNA. Evidence that the remaining b a c t e r i a were able to metabolize and i n c o r p o r a t e 5-FU i s given i n s e c t i o n s 2.6 and 2 .7 . At one stage of t h i s work two pounds of 5-FU t r e a t e d E. c o l i B c e l l s were g r a c i o u s l y provided by P r o f e s s o r Ivan K a i s e r at the U n i v e r s i t y o f Wyoming. His kindness i s g r e a t l y appre-c i a t e d . - 64 -2.3 I s o l a t i o n o f 5SrRNA The i s o l a t i o n p r o c e d u r e s f o r 5SrRNA from u n t r e a t e d and 5-FU t r e a t e d E. c o l i B c e l l s were i d e n t i c a l . The water s o l u b l e RNA (sRNA) was e x t r a c t e d from the b a c t e r i a w i t h an e m u l s i o n t h a t c o n s i s t e d o f e q u a l volumes o f water and p h e n o l . The p h e n o l f a c i l i t a t e s e x t r a c t i o n o f the sRNA by i n c r e a s i n g the p o r o s i t y of the c e l l s through the e x t r a c t i o n o f l i p i d s and p r o t e i n s from the c e l l membrane. Phenol a l s o d e n a t u r e s r i b o n u c l e a s e s t o p r e -vent e n z y m a t i c d e g r a d a t i o n o f the RNA. The combined e f f e c t s o f the p h e n o l a l l o w i n t a c t tRNA, 5SrRNA, and a s m a l l amount of the l a r g e r rRNA t o l e a k out o f the c e l l s and d i s s o l v e i n the aque-ous phase of the w a t e r - p h e n o l e m u l s i o n . DNA and most of the rRNA remain i n s i d e the c e l l membrane due t o t h e i r l a r g e r s i z e . The aqueous phase can be s e p a r a t e d from p h e n o l and c e l l u l a r d e b r i s by c e n t r i f u g a t i o n . The sRNA i s o l a t e d by the above t e c h n i q u e c o n s i s t s o f about 8% rRNA, 86% tRNA, and . 6% 5SrRNA (see F i g u r e 2.2). S i n c e t h i s m i x t u r e c o n s i s t s of t h r e e m o l e c u l e s which d i f f e r i n s i z e and shape, the 5SrRNA was c o n v e n i e n t l y s e p a r a t e d from the o t h e r two RNA s p e c i e s by g e l f i l t r a t i o n chromatography u s i n g Sephadex G-100 or G-75 g e l s . The f i n a l s t e p o f the i s o l a t i o n p rocedure was t o d e s a l t the 5SrRNA on a Sephadex G-25 column and then l y o p h i l i z e i t . The r e s u l t i n g f r e e z e d r i e d powder was then s t o r e d a t -20°C. - 65 -The d e t a i l e d i s o l a t i o n procedures are o u t l i n e d below i n f i v e s t e p s . Steps I, I I , and I I I were designed by Dr. Gordon Tener of the U n i v e r s i t y of B r i t i s h Columbia Bio c h e m i s t r y De-partment. Gel f i l t r a t i o n procedures s i m i l a r to step IV have been employed by others (2). I t was p o s s i b l e to o b t a i n between 10 and 20 m i l l i g r a m s of 5SrRNA per 100 grams of E. c o l i B c e l l s . Step I. P r e p a r a t i o n of B u f f e r s and Columns The t r i s b u f f e r r e f e r r e d to i n steps I-IV was prepared with d e i o n i z e d water and contained 0.01 molar t r i s - ( h y d r o x y -methyl)-methylamine-hydrogen c h l o r i d e ( t r i s - H C l ) a d j u s t e d to pH 7.5 using a s o l u t i o n of sodium hydroxide. To assure the presence of a n a t i v e c o n c e n t r a t i o n of magnesium ions the b u f f e r a l s o contained 10 m i l l i m o l a r magnesium c h l o r i d e . Fresh t r i s . b u f f e r was prepared every two days to prevent b a c t e r i a l growth in the b u f f e r and on the v a r i o u s columns. A d i e t h y l amino c e l l u l o s e (DEAE-cellulose) column was pre-pared p r i o r to the i s o l a t i o n of sRNA. For each 100 grams of E. c o l i B c e l l s to be used i n step II 40 grams of Whatman DEAE-c e l l u l o s e (0.86 m i l l i e q u i v a l e n t s per gram) was mixed with 4 l i t e r s of d e i o n i z e d water. To f a c i l i t a t e a convenient flow rate f i n e s were removed by a l l o w i n g the D E A E - c e l l u l o s e to s e t -t l e f o r about 30 minutes. Then most of the water was decanted o f f and the process repeated a second time. The D E A E - c e l l u l o s e was then poured i n t o a column which was at l e a s t 2.5 c e n t i -meters i n diameter. T h i s column was washed with t r i s b u f f e r - 66 -t h a t c o n t a i n e d 1 molar sodium c h l o r i d e u n t i l the &260 o f the e l u e n t was l e s s than 0.05. Then i t was e q u i l i b r a t e d w i t h a t l e a s t 3 volumes o f t r i s b u f f e r c o n t a i n i n g 0.3 molar sodium c h l o r i d e . Sephadex G-100 (or G-75) columns were p r e p a r e d a c c o r d i n g to the m a n u f a c t u r e r ' s s p e c i f i c a t i o n ( 3 ) . For the i s o l a t i o n o f 5SrRNA used i n 1 9F-nmr e x p e r i m e n t s the g e l s were poured i n t o 5 x 90 c e n t i m e t e r columns. Longer narrower columns (2 x 180 c e n t i m e t e r s ) were l a t e r adapted f o r l a s e r Raman s t u d i e s . The e l u e n t i n a l l c a s es was t r i s b u f f e r c o n t a i n i n g 1 molar sodium c h l o r i d e . S tep I I . E x t r a c t i o n o f sRNA from E. c o l i B c e l l s To each 100 grams o f f r o z e n E. c o l i B c e l l s 300 m i l l i l i t e r s o f t r i s b u f f e r and an e q u a l volume o f water s a t u r a t e d p h e n o l were added. The r e s u l t i n g w a t e r - p h e n o l e m u l s i o n was mixed w i t h the b a c t e r i a l c e l l s f o r a t l e a s t 30 minutes and then c e n t r i -fuged a t 10,000 g f o r 15 minutes on a S o r v a l RC2-B r e f r i g e r a t e d c e n t r i f u g e . C e n t r i f u g a t i o n f a c i l i t a t e d the s e p a r a t i o n o f the aqueous phase from the p h e n o l and the c e l l u l a r d e b r i s . The top aqueous phase was removed by s u c t i o n and another 300 m i l l i l i t e r s o f t r i s b u f f e r was added t o the r e m a i n i n g p h e n o l - c e l l d e b r i s m i x t u r e . A f t e r m i x i n g and c e n t r i f u g a t i o n t h i s second aqueous phase was combined w i t h the f i r s t and 0.1 volumes o f 2 molar p o t a s s i u m a c e t a t e (pH 4.5) was added. The sRNA was then p r e -c i p i t a t e d by the a d d i t i o n o f 2.5 volumes o f 95% e t h a n o l - 67 -p r e - c o o l e d t o -20 d e g r e e s c e n t i g r a d e . The p r e c i p i t a t e was a l -l o w e d t o f l o c c u l a t e and s e t t l e o v e r n i g h t i n t h e f r e e z e r . The n e x t d a y most o f t h e e t h a n o l - w a t e r s o l u t i o n was d e c a n t e d o f f and t h e r e m a i n i n g RNA p r e c i p i t a t e - e t h a n o l m i x t u r e was c e n t r i -f u g e d a t 10,000 g f o r 10 m i n u t e s . The r e s u l t i n g sRNA p e l l e t was washed t w i c e w i t h c o l d 95% e t h a n o l . A f t e r t h e s e c o n d w a s h i n g and c e n t r i f u g a t i o n t h e RNA was t h o r o u g h l y d r a i n e d o f t h e e t h a n o l . S t e p I I I . A t t a c h m e n t o f sRNA t o a D E A E - c e l l u l o s e c o l u m n and  s u b s e q u e n t w a s h i n g o f t h e RNA The sRNA f r o m S t e p I I was d i s s o l v e d i n a m i n i m a l amount o f t r i s b u f f e r c o n t a i n i n g 0.3 m o l a r s o d i u m c h l o r i d e ( a p p r o x i m a t e l y 50 m i l l i l i t e r s f o r sRNA f r o m 100 grams o f f r o z e n E. c o l i B c e l l s ) . The s o l u t i o n was t h e n c e n t r i f u g e d a t 10,000 g f o r 15 m i n u t e s t o remove a s m a l l amount o f i n s o l u b l e d e b r i s . The sRNA s o l u t i o n was a p p l i e d t o t h e D E A E - c e l l u l o s e c o l u m n w h i c h was e q u i l i b r a t i n g w i t h t r i s b u f f e r c o n t a i n i n g 0.3 m o l a r s o d i u m c h l o r i d e ( s ee s t e p I ) . A t t h i s s a l t c o n c e n t r a t i o n t h e sRNA w i l l a t t a c h t o t h e DEAE a n i o n e x c h a n g e r and c a n be washed w i t h t r i s b u f f e r c o n t a i n i n g 0.3 m o l a r s o d i u m c h l o r i d e . T h i s f a c i l i t a t e s r e m o v a l o f c a r b o h y d r a t e s , p h e n o l , m o n o n u c l e o t i d e s and p r o t e i n s w h i c h do n o t b i n d i n t h e p r e s e n c e o f 0.3 m o l a r s o d i u m c h l o r i d e . W a s h i n g i s c o n t i n u e d u n t i l t h e A 2gQ r e d u c e s t o a minimum ( a b o u t 500 m i l l i l i t e r s ) . The sRNA was t h e n e l u t e d f r o m t h e c o l u m n w i t h t r i s b u f f e r c o n -t a i n i n g 1 m o l a r s o d i u m c h l o r i d e . The e l u e n t w i t h an ^260 9 ^ e a t — e r t h a n 0.5 was c o l l e c t e d and 20 m i l l i l i t e r s o f 0.1 m o l a r mag-n e s i u m c h l o r i d e was a d d e d . The sRNA was t h e n p r e c i p i t a t e d w i t h - 68 -2.5 volumes o f 95% e t h a n o l p r e - c o o l e d t o -20 degrees c e n t i g r a d e . The "washed" sRNA was a l l o w e d t o f l o c c u l a t e and s e t t l e o v e r n i g h t a t -20 degrees c e n t i g r a d e . D e c a n t a t i o n and c e n t r i f u g a t i o n (10,000 g f o r 10 minutes) was then used t o o b t a i n the sRNA p e l l e t . Step IV. I s o l a t i o n o f 5SrRNA from sRNA The s e p a r a t i o n o f 5sRNA from tRNA and rRNA was a c c o m p l i s h e d by g e l f i l t r a t i o n chromatography on Sephadex G-100 or G-75 columns. An e l u t i o n p r o f i l e o f sRNA from a Sephadex G-75 c o l -umn i s shown i n F i g u r e 2.2. A c c o r d i n g t o t h i s p r o f i l e sRNA from s t e p I I I c o n s i s t s o f about 86% tRNA, 6% 5SrRNA, and 8% rRNA. T h i s i s i n agreement w i t h the r e l a t i v e p r o p o r t i o n s ob-t a i n e d by o t h e r s ( 2 ) . Sephadex G-75 (or G-100) was p r e p a r e d a c c o r d i n g t o the m a n u f a c t u r e r ' s s p e c i f i c a t i o n s and b e f o r e u s i n g the columns were e q u i l i b r a t e d w i t h a t l e a s t 2 volumes of t r i s b u f f e r c o n t a i n i n g 1 molar sodium c h l o r i d e . The b e s t s e p a r a t i o n was o b t a i n e d w i t h l o n g narrow columns. The 5SrRNA p r e p a r e d f o r the l a s e r Raman ex p e r i m e n t s was p u r i f i e d u s i n g 2 x 190 c e n t i m e t e r columns. The 5SrRNA f o r the l 9F-nmr e x p e r i m e n t s , where s u b s t a n t i a l l y l a r g e r sample amounts were r e q u i r e d , was p u r i f i e d on s h o r t e r and wider columns (about 5 x 90 c e n t i m e t e r s ) . The l a t t e r samples c o n t a i n e d s l i g h t l y l a r g e r t r a c e s of tRNA c o n t a m i n a t i o n as judged by 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 (see s e c t i o n 2.5). E l u t i o n p r o f i l e s were o b t a i n e d w i t h an LKB 8300 U v i c o r d I I connected t o a F i s h e r R e c o r d a l l S e r i e s 5000 c h a r t r e c o r d e r . 69 -. 9 . 8 . 7 . 6 ° 51 CM < .4 .3 .2 .1 r R N A i E l u t i o n V o l u m e Figure 2.2. A complete e l u t i o n p r o f i l e of sRNA a p p l i e d to a Sephadex G-100 column (5 x 90 c e n t i m e t e r s ) . The l a r g e s t mole-c u l a r weight component, rRNA, e l u t e s at the column's v o i d volume. The tRNA, which i s the s m a l l e s t , i s r e t a i n e d longest on the column. The 5SrRNA, which i s intermediate i n s i z e , e l u t e s j u s t before the tRNA peak. According to t h i s e l u t i o n p r o f i l e sRNA c o n s i s t s of about 86% tRNA, 8% rRNA, and 6% 5 S r RNA. - 70 -In g e n e r a l the Sephadex column was overloaded with r e s p e c t to tRNA and rRNA. T h i s was done to achieve l a r g e r y i e l d s of 5SrRNA per run. A s i n g l e run on a 2 x 190 centimeter column r e q u i r e d at l e a s t 8 hours. The sRNA from about 50 grams of c e l l s was the maximum that c o u l d be used f o r a s i n g l e run w i t h -out completely d e s t r o y i n g peak r e s o l u t i o n . The e l u t i o n p r o f i l e f o r sRNA from about 25 grams of c e l l s e l u t e d from a 2 x 190 centimeter column i s shown in F i g u r e 2.3. The e l u e n t was c o l -l e c t e d using a G i l f o r d micro fractionator and a l i q u o t s from the center most peak, corresponding to 5SrRNA, were c o l l e c t e d and p r e c i p i t a t e d by the a d d i t i o n of 0.1 volumes of 0.1 molar mag-nesium c h l o r i d e f o l l o wed by the a d d i t i o n of 2.5 volumes of 95% e t h a n o l cooled to -20 degrees c e n t i g r a d e . A f t e r the p r e c i p i -t a t e was allowed to s e t t l e f o r at l e a s t one day i t was c o l l e c t e d by d e c a n t a t i o n and c e n t r i f u g a t i o n at 10,000 g f o r 10 minutes. The r e s u l t i n g p e l l e t , c o n s i s t i n g predominantly of 5SrRNA, was d i s s o l v e d i n a minimal amount of t r i s b u f f e r c o n t a i n i n g 1 molar sodium c h l o r i d e and r e a p p l i e d to the Sephadex column.;. An e l u -t i o n p r o f i l e of a second run on t h i s column i s shown i n F i g u r e 2.4. The f r a c t i o n s corresponding to 5SrRNA were again c o l l e c t e d and p r e c i p i t a t e d as mentioned above. A f t e r the second run the 5SrRNA sample was judged pure according to Sephadex chromato-graphy. The e l u t i o n p r o f i l e of p u r i f i e d 5SrRNA run on the same Sephadex column i s shown i n F i g u r e 2.5. Superimposed with i t i s an e l u t i o n p r o f i l e of tRNA. - 71 -tRNA 5SrRNA rRNA Tube Number F i g u r e 2.3. The e l u t i o n p r o f i l e f o r sRNA from a p p r o x i m a t e l y grams o f E. c o l i B c e l l s . S e p a r a t i o n i s on a Sephadex G-75 column (2 x 180 c e n t i m e t e r s ) . The i n t e r m e d i a t e peak, c o r r e s p o n d i n g t o 5SrRNA, was c o l l e c t e d and p r e c i p i t a t e d w i t h 95% e t h a n o l p r e - c o o l e d t o -20° C as d e s c r i b e d i n the t e x t . - 72 -d> o c CO E CO c CO 0 + 2 0 + 4 0 + 6 0 + 8 0 + 5S r^ R NA rRNA 1 0 0 J - H - r + | i II i | i i n | II I 111 11 11 i i n I i| i 11 | i h + F F f f f 5 0 4 0 3 0 2 0 Tube Number Figure 2.4. An e l u t i o n p r o f i l e of the 5SrRNA component ob-tain e d from Fi g u r e 2.3. A f t e r t h i s second pass over the column the sample was judged homogeneous according to Sephadex G-75 chromatography (see Fig u r e 2.5). - 73 -0 O c CO E CO c CO 0 + 2 0 + 4 0 + 6 0 8 0 + 5SrRNA tRNA 1 0 0 1 1 ' M 11 M I I I I 11 I I I I 11 | I l I 1 I I 11 I 11 l 11 | I I i 11 l 11 I 5 0 4 0 3 0 Tube Number 2 0 Figure 2.5. A demonstration of 5SrRNA homogeneity according to. Sephadex G-75 chromatography. The s o l i d l i n e d p r o f i l e r e p r e -sents a 5SrRNA sample. The dotted l i n e r e p r e s e n t s a tRNA sample which was run independently. These p r o f i l e s were both obtained on the same column used i n F i g u r e s 2.3 and 2.4. - 74 -Step V. D e s a l t i n g and L y o p h i l i z a t i o n of 5SrRNA The 5SrRNA p e l l e t from step IV was d i s s o l v e d i n a minimal amount of d e i o n i z e d water and a p p l i e d to a Sephadex G-25 column (4). The e l u e n t was monitored with the LKB U v i c o r d II and f r a c -t i o n s absorbing at 260 m i l l i m i c r o n s were c o l l e c t e d . The e l u e n t was poured i n t o a l y o p h i l i z a t i o n f l a s k and f r o z e n by immersing the s p i n n i n g f l a s k i n an N-propanol-dry i c e bath. A f t e r the s o l u t i o n was f r o z e n the f l a s k was attached to a V i r t u s U n i t r a p II l y o p h i l i z a t i o n apparatus. The r e s u l t i n g pure f r e e z e - d r i e d 5SrRNA was weighed and s t o r e d at -20 degrees c e n t i g r a d e . - 75 -2.4 Separation of Normal 5SrRNA from FU-5SrRNA The 5SrRNA i s o l a t e d from E. c o l i B c e l l s grown i n the pre-sence of 5-FU c o n s i s t e d of a mixture of normal u n f l u o r i n a t e d 5SrRNA and , FU-5SrRNA. Ivan K a i s e r has shown that both un-f l u o r i n a t e d n a t i v e tRNA and 5SrRNA can be separated from the 5-FU c o n t a i n i n g molecules by DEA E - c e l l u l o s e chromatography (5-6). A r e d u c t i o n of the pK g for 5-FU (8.15 compared to 9.45 f o r nor-mal u r a c i l ) means that at a b u f f e r pH of 8.9 most of the 5-FU bases i n 5-FU c o n t a i n i n g RNA w i l l be deprotonated at the N-3 p o s i t i o n of the 5-FU base. T h i s p r o v i d e s a g r e a t e r a f f i n i t y of the FU-RNA f o r the D E A E - c e l l u l o s e column. T h e r e f o r e , when a mixture of normal 5SrRNA and , FU-5SrRNA i s e l u t e d with an up-wardly concave s a l t g r a d i e n t of i n c r e a s i n g sodium c h l o r i d e con-c e n t r a t i o n the FU-5SrRNA i s more s t r o n g l y r e t a i n e d on the column and e l u t e s a f t e r the normal 5SrRNA. The r e s u l t i n g e n r i c h e d FU-5SrRNA has been shown to e x h i b i t about 83% replacement of the U base by 5-FU (6). Most FU-5SrRNA enrichment experiments were performed with a 0.9 x 40 centimeter Whatman DE 32 column according to the pro-cedure of K a i s e r (6). The DEA E - c e l l u l o s e was p r e c y c l e d as pre-s c r i b e d by the manufacturer (7). A f t e r the p r e c y c l e d m a t e r i a l was poured i n t o the column i t was g e n e r a l l y washed with 3 v o l -umes of high s a l t b u f f e r (pH 8.9). The high s a l t b u f f e r con-t a i n e d 0.02 molar t r i s (pH 8.9), 0.01 molar magnesium c h l o r i d e and 1 molar sodium c h l o r i d e . Then i t w a s - e q u i l i b r a t e d with 3 - 76 -volumes of 0.02 molar t r i s b u f f e r (pH 8.9) c o n t a i n i n g 0.3 molar sodium c h l o r i d e and 0.01 molar magnesium c h l o r i d e (low s a l t b u f f e r ) . The 5SrRNA sample, d i s s o l v e d i n low s a l t b u f f e r , was a p p l i e d to the column and allowed to -wash i n with a smal l amount of the same b u f f e r . The maximum amount of 5SrRNA that c o u l d be a p p l i e d to the column was about 200 &260 u n i t s • A schematic diagram of the g r a d i e n t maker used f o r most enrichment experiments i s shown i n F i g u r e 2.6. The diameter of the mixing r e s e r v o i r was 5.7 centimeters while the smaller r e -s e r v o i r had a diameter of 3.0 c e n t i m e t e r s . To the mixing r e s e r -v o i r 235 m i l l i l i t e r s of the low s a l t b u f f e r (0.3 molar sodium c h l o r i d e ) mentioned above and a s t i r bar were added. The smaller r e s e r v o i r was f i l l e d with about 70 m i l l i l i t e r s of the same buf-fe r c o n t a i n i n g 0.7 molar sodium c h l o r i d e . The two r e s e r v o i r s were i n t e r c o n n e c t e d by means of p o l y e t h y l e n e tubing i n s e r t e d i n t o t h e i r bases. The shape of the g r a d i e n t was estimated a c c o r d i n g to the formula d e r i v e d i n F i g u r e 2.6. A G i l f o r d m i c r o f r a c t i o n a t o r was used to c o l l e c t the e l u e n t and s e p a r a t i o n was monitored by measurement of the &260 e i t n e r by an LKB Uvi c o r d II connected to a F i s h e r R e c o r d a l l S e r i e s 5000 recorder or as i n d i v i d u a l f r a c t i o n s on a G i l f o r d 240 spec-trometer. The A 2 8 0 : A 2 6 0 r a 1 - i 0 w a s u s e c> a s a n i n d i c a t i o n of 5-FU enrichment s i n c e the , FU-5SrRNA has an i n c r e a s e d value f o r t h i s r a t i o (approximately 0.55 as compared to 0.52) (6). A t y p i c a l e l u t i o n p r o f i l e i s shown i n F i g u r e 2.7. a - 77 -High Salt Mix ing Reservoir !2) Reservoir( i) T h valve l - t h e i n i t i a l d i s t a n c e b e t w e e n t h e s u r f a c e o f t h e b u f f e r s o l u t i o n a n d t h e b o t t o m o f t h e r e s e r v o i r , r ^ - r a d i u s o f t h e h i g h s a l t c y c l i n d r i c a l r e s e r v o i r . 12 - r a d i u s o f t h e c y c l i n d r i c a l m i x i n g r e s e r v o i r . C i ( i ) - i n i t i a l c o n c e n t r a t i o n o f b u f f e r i n t h e h i g h s a l t r e s e r v o i r . C 2 ( i ) - i n i t i a l c o n c e n t r a t i o n o f l o w s a l t b u f f e r i n t h e m i x i n g r e s e r v o i r . _ V T 2 - i n i t i a l v o l u m e o f l o w s a l t b u f f e r i n t h e m i x i n g r e s e r v o i r : V T 2 = S-Ilr, h - c h a n g e i n s u r f a c e h e i g h t o f t h e b u f f e r r e s u l t i n g f r o m t h e f l o w o f b u f f e r i n t h e h i g h s a l t r e s e r v o i r . V i ( h ) - t h e v o l u m e o f b u f f e r i n t h e h i g h s a l t r e s e r v o i r a s a f u n c t i o n o f h : V n (h) = h l l r , " V , ( h ) - t i t e v o l u m e b u f f e r i n t h e l o w s a l t r e s e r v o i r a s a f u n c t i o n o f h : V , (h) = h n r _ F r o m t h e a b o v e p a r a m e t e r s t h e s a l t c o n c e n t r a t i o n o f t h e b u f f e r i n t h e m i x i n g r e s e r v o i r (C2<h)) a s a f u n c t i o n o f h i s d e r i v e d a s f o l l o w s : = V 1 ( h ) r j C 1 ( i ) ] +rV T 2 - V2W2£c2{i)2 C , ( h ) V-^h) rvT 2 - v2(h>] C VT2 " V2<h>D D V i ) ! ] + C c2 ( i>I] S u b s t i t u t i n g t h e f o l l o w i n g e q u a t i o n s i n t o e q u a t i o n 2: V-^h) = h H r x 2 V 2 ( h ) = h n r 2 2 "T2 «.nr. (1) (2) (3) (4) (5) o n e a r r i v e s a t t h e f i n a l e x p r e s s i o n f o r C 2 ( h ) , C 2 ( h ) T h i s i s g i v e n b y e q u a t i o n 6. (6) Figure 2.6. A schematic s i d e view of the c y c l i n d r i c a l r e s e r -v o i r s used to generate concave upward g r a d i e n t s of i n c r e a s i n g s a l t c o n c e n t r a t i o n . Such g r a d i e n t s were used to separate N-5SrRNA from FU-5SrRNA on DEAE columns (see text) . A l s o i n -cluded i s a d e r i v a t i o n of the s a l t c o n c e n t r a t i o n of the mixing r e s e r v o i r (C 2(h)) as a f u n c t i o n of the change i n surface height (h) which r e s u l t s from the flow of b u f f e r to the DEAE column. - 78 -'260 NaCl '(molar) FU-5SrRNA A 2 9 P i ' i i | i i i i f*f i i i | i i i i | i i i i | i i i i | i i t i | i i i i | i i i i 10 20 30 Tube 40 50 Number 60 70 80 F i g u r e 2.7. The s e p a r a t i o n of N-5SrRNA from FU-5SrRNA by DEAE-c e l l u l o s e chromatography. The s o l i d dots represent the e l u t i o n p r o f i l e of the RNA monitored by A260 * The s o l i d diamonds i n -d i c a t e A280 : A260 r a t i o s and the c i r c l e s r epresent the estimated NaCl c o n c e n t r a t i o n c a l c u l a t e d a c cording to the equation shown in Figure 2.6. The column dimensions were 0.9 x 40 centimeters and the e l u e n t per tube was about 3.4 m i l l i l i t e r s . - 79 -The f r a c t i o n corresponding to e n r i c h e d ,FU-5SrRNA was p r e c i p i t a t e d with 95% e t h a n o l cooled to -20 degrees c e n t i g r a d e a f t e r the a d d i t i o n of 0 .1 volumes of 0 .1 molar magnesium c h l o r -i d e . The p r e c i p i t a t e was allowed to s e t t l e o v e r n i g h t at -20 degrees c e n t i g r a d e . Then a f t e r most of the s o l u t i o n was de-canted i t was c e n t r i f u g e d a t 10,000 g f o r about 10 minutes. The e n r i c h e d FU-5SrRNA p e l l e t was then d r a i n e d and d i s s o l v e d i n a minimal amount of d e i o n i z e d water. D e s a l t i n g and f r e e z e -d r y i n g of the sample was according to the procedure o u t l i n e d i n Step V of S e c t i o n 2 .3 . The purpose of t h i s 5 - f l u o r o u r a c i l enrichment procedure was to improve the 1 9F-nmr s i g n a l to noise r a t i o by removing u n f l u o r i n a t e d 5SrRNA. T h i s reduced the a c q u i s i t i o n time r e -q u i r e d to o b t a i n FU-5SrRNA 1 9F-nmr s p e c t r a . Both the broadness of the DEAE p r o f i l e and the A 280 / / A 260 r a f c :'- o s s h o w n i - n F i g u r e 2.7 suggest h e t e r o g e n e i t y with r e s p e c t to number of f l u o r o -u r a c i l s per molecule. However each of the f l u o r i n e resonances i s r e a l l y the sum of the f l u o r i n e l a b e l (or l a b e l s ) from a s p e c i f i c r e g i o n of each 5SrRNA molecule contained i n the nmr sample tube. Incomplete f l u o r i n a t i o n of some of the FU-5SrRNA molecules w i l l have some e f f e c t on the i n t e n s i t y but not the p o s i t i o n or T^ of resonances due to the absence of f l u o r i n e l a b e l s i n s p e c i f i c molecules. Consequently h e t e r o g e n e i t y with r e s p e c t to 5 - f l u o r o u r a c i l l a b e l i n g i n FU-5SrRNA should not a f f e c t the s t r u c t u r a l and c o n f o r m a t i o n a l i n t e r p r e t a t i o n of the 1 9F-nmr r e s u l t s . - 80 -2.5 Sample P u r i t y by Polyacrylamide Gel E l e c t r o p h o r e s i s The p u r i t y of 5SrRNA samples was p e r i o d i c a l l y checked by polyacrylamide g e l e l e c t r o p h o r e s i s . A photograph of a sample g e l i s shown i n F i g u r e 2.8. As shown tRNA samples were run c o n c u r r e n t l y as standards to b e t t e r d i s c r i m i n a t e the p o s i t i o n of 5SrRNA. The band c h a r a c t e r i z i n g 5SrRNA i s a c t u a l l y two c l o s e l y p o s i t i o n e d bands r e p r e s e n t i n g the A and B conformations of t h i s RNA s p e c i e s (8). A l l samples i n d i c a t e d s l i g h t t r a c e s of tRNA contamination. The amount of contamination was s m a l l e s t f o r samples obtained from the longer-narrower columns (2 x 190 centimeters i n s t e a d of the 5 x 90 c e n t i m e t e r s ) . The homogeneity of SSrRNA from the longer columns was judged to be much grea t e r than 95%. The e l e c t r o p h o r e s i s procedure o u t l i n e d below i s d i v i d e d i n t o 4 steps (9). Step I. P r e p a r a t i o n of the E l e c t r o d e B u f f e r The e l e c t r o d e b u f f e r c o n s i s t e d of 20 m i l l i m o l a r t r i s - a c e -t a t e , pH 8.0, prepared with d e i o n i z e d water and c o n t a i n i n g 1 m i l l i m o l a r EDTA and 4 molar urea. U s u a l l y 1 l i t e r of t h i s buf-f e r was s u f f i c i e n t f o r each experiment. Step I I . P r e p a r a t i o n of Polyacrylamide Gel Slabs F i r s t , 50 . m i l l i l i t e r s of acrylamide s o l u t i o n was prepared. T h i s s o l u t i o n c o n s i s t e d of 10% acrylamide and 0.5% b i s a c r y l a m i d e prepared with the e l e c t r o d e b u f f e r of step I. The s o l u t i o n was then mixed with 0.025 m i l l i l i t e r s of N,N,N',N'-tetramethyethylene - 81 -diamine and 0.2 m i l l i l i t e r s of f r e s h l y prepared 10% ammonium s u l f a t e . T h i s mixture was immediately placed i n the s l a b making device and allowed to s o l i d i f y . A f t e r s o l i d i f i c a t i o n i t was pre-run f o r about 3 hours with 20 m i l l i a m p e r e s of c u r r e n t . Step I I I . P r e p a r a t i o n of RNA Samples f o r E l e c t r o p h o r e s i s RNA samples were d i s s o l v e d i n a 20% sucrose s o l u t i o n pre-pared with the e l e c t r o d e b u f f e r (step I) and c o n t a i n i n g 0.05% bromophenol b l u e . U s u a l l y a 1 m i l l i g r a m RNA per m i l l i l i t e r s o l u t i o n was prepared by mixing 0.1 m i l l i g r a m s of RNA with 0.1 m i l l i l i t e r s of t h i s s o l u t i o n . Then the s o l u t i o n was heated to 65 degrees c e n t i g r a d e f o r 1 minute. A f t e r pre-running of the poly a c r y l a m i d e s l a b 10 m i c r o l i t e r s o f t h i s RNA s o l u t i o n was c a r e f u l l y a p p l i e d to a p p r o p r i a t e s l o t s on the s l a b . E l e c t r o -p h o r e s i s was c a r r i e d out, at room temperature, f o r approximately 4 hours with a c u r r e n t of 15-20 m i l l i a m p e r e s . The m i g r a t i o n of the bromophenol blue dye to the end of the s l a b i n d i c a t e d com-p l e t i o n . Step IV. P r e p a r a t i o n of the RNA S t a i n The RNA s t a i n i n g s o l u t i o n c o n s i s t e d o f 0.2% methylene blue in d e i o n i z e d water that contained 0.02 molar sodium acetate and 0.02 molar a c e t i c a c i d . The g e l s l a b was immersed f o r about 30 minutes i n t h i s s o l u t i o n and then washed o v e r n i g h t with c o l d tap water. - 8 2 -1 > u - F U - N- F U - N-pS—RNA SS-RNA 5S-RNA t-RNA t-RNA Figure 2.8. Sample purity was p e r i o d i c a l l y checked by poly-acrylamide gel electrophoresis. As indicated tRNA samples were run concurrently to better discriminate the position of 5SrRNA. - 83 -2.6 V e r i f i c a t i o n of 5-FU I n c o r p o r a t i o n i n t o E. c o l i RNA The a d d i t i o n of 5-FU to media c o n t a i n i n g a c t i v e l y growing E. c o l i B c e l l s caused c e l l u l a r d e s t r u c t i o n and the c e s s a t i o n of c e l l d i v i s i o n . T h i s prompted concern about the extent of 5-FU i n c o r p o r a t i o n i n t o the b a c t e r i a ' s RNA. In order to d e t e r -mine whether 5-FU was being a c t i v e l y metabolized by the remain-ing E. c o l i B c e l l s an experiment was devised using 5-FU l a b e l e d with carbon-14 at the 2-carbon p o s i t i o n . Since s m a l l q u a n t i t i e s of c e l l s were employed the r a d i o a c t i v i t y i n i s o l a t e d tRNA was used to evaluate the degree of 5-FU i n c o r p o r a t i o n . One l i t e r of s t e r i l i z e d media (see s e c t i o n 2.2) was i n -o c u l a t e d with 10 m i l l i l i t e r s of an E. c o l i B c u l t u r e which had been growing o v e r n i g h t at room temperature on a shaker. A f t e r i n o c u l a t i o n the f l a s k was placed on a shaker, at ambient tem-pe r a t u r e , u n t i l t u r b i d i t y measurements i n d i c a t e d e a r l y expo-n e n t i a l growth ( Aggg - 0.3). Then 25 m i l l i g r a m s of 5-FU was added. A f t e r about 5 minutes 20 m i c r o c u r i e s of carbon-14 l a -beled 5-FU with a s p e c i f i c a c t i v i t y equal to 10.26 m i l l i c u r i e s per m i l l i m o l e was added. The f l a s k remained on the shaker for 3 hours. The c e l l s were harvested by c e n t r i f u g a t i o n (10,000 g fo r 10 minutes) and the y i e l d was about one gram of c e l l s (wet weight). The sRNA was i s o l a t e d a c c o r d i n g to the procedure out-l i n e d i n s e c t i o n 2.3, step I I . The tRNA was separated from the rRNA on a s m a l l Sephadex G-100 column (about 0.5 x 40 c e n t i -meters). F r a c t i o n s were c o l l e c t e d with a G i l s o n m i c r o f r a c t i o n -ator and the &260 w a s m o n ; ' - t o r e c ' o n the G i l f o r d 240 spectrometer. - 84 -Measurement of r a d i o a c t i v i t y was obtained on the departmental s c i n t i l l a t i o n counter. F r a c t i o n s of 1.5 m i l l i l i t e r s each were c o l l e c t e d on the m i c r o f r a c t i o n a t o r . A l i q u o t s of 0.5 m i l l i l i t e r s were a c c u r a t e l y withdrawn from a l t e r n a t e f r a c t i o n s and placed i n s c i n t i l l a t i o n v i a l s which contained 5 m i l l i l i t e r s of F i s h e r S c i n t i v e r s e s c i n t i l l a t i o n f l u i d . Then the counts per minute for each v i a l was obtained on the s c i n t i l l a t i o n counter. Pre-v i o u s to t h i s experiment a standard curve was determined from samples of known d i s i n t e g r a t i o n s per minute (D.P.M.). This curve i s shown i n Figu r e 2 .9 . The D.P.M. for each sample was then determined from i t s counting e f f i c i e n c y obtained from F i g -ure 2 .9 . The Sephadex G-100 e l u t i o n p r o f i l e and corresponding D.P.M. measurements are recorded i n Fig u r e 2 .10. The area under t h i s curve, which corresponds to tRNA, rep r e s e n t s 18.9 u n i t s o r -5 0.86 m i l l i g r a m s (3.44 x 10 m i l l i m o l e s ) assuming that h i g h l y p u r i f i e d tRNA has an &26Q °^ 7.45 per micromole phosphorus (or 22 A 2gQ u n i t s per m i l l i g r a m tRNA) (10) . The area underneath the r a d i o a c t i v i t y curve, a s s o c i a t e d with the tRNA e l u t i o n p r o f i l e , -8 corresponds to about 93,108 D.P.M. or 4.19 x 10" curies. Given that the s p e c i f i c a c t i v i t y of the r a d i o a c t i v e l y l a b e l e d 5-FU was 10.26 m i l l i c u r i e s per m i l l i m o l e the r a d i o a c t i v e 5-FU i n 0.86 _5 m i l l i g r a m s of tRNA (3.44 x 10 m i l l i m o l e s ) was determined to — fi be equal to 4.09 x 10 m i l l i m o l e s . Since 20 m i c r o c u r i e s of _3 l a b e l e d 5-FU (1.94 x 10 m i l l i m o l e s ) and 25 m i l l i g r a m s of un-la b e l e d 5-FU (0.2 m i l l i m o l e s ) were added to the growing E. c o l i - 85 -H i 1 1 1 1 + f 1 2 3 4 5 6 7 8 § Ratio Figure 2.9. A c a l i b r a t i o n curve of counting e f f i c i e n c y versus c h a n n e l - r a t i o s f o r samples with known D.P.M. v a l u e s . T h i s curve was used to determine the D.P.M. values f o r unknowns by e x t r a p o l a t i o n of t h e i r counting e f f i c i e n c y from the channel-r a t i o s of each sample using t h i s curve. - 86 -Tube Number Figure 2.10. A Sephadex G-100 chromatography of sRNA which c o n t a i n s l h C l a b e l e d 5-FU r e s i d u e s . The dimensions of the column were about 1.5 x 40 c e n t i m e t e r s . The s o l i d l i n e con-n e c t i n g the s o l i d dots i n d i c a t e s the A260 measurements of the e l u e n t from i n d i v i d u a l f r a c t i o n s . Each tube contained 1.5 m i l l i l i t e r s of e l u e n t . For the r a d i o a c t i v i t y measurements (the dotted l i n e connected by hollow c i r c l e s ) 0.5 m i l l i l i t e r s of e l u e n t were withdrawn from a l t e r n a t e f r a c t i o n s and placed in s c i n t i l l a t i o n v i a l s c o n t a i n i n g F i s h e r S c i n t i v e r s e s c i n t i l -l a t i o n f l u i d . C h a n n e l - r a t i o s f o r the samples were obtained on the departmental s c i n t i l l a t i o n counter. Counting e f f i c i e n -c i e s were obtained from these r a t i o s by e x t r a p o l a t i o n using Figure 2.9. From the e f f i c i e n c i e s the D.P.M. values f o r the samples were computed. - 87 -B c e l l s the r a t i o of l a b e l e d to unlabeled 5-FU was 102.6. Mul-t i p l i c a t i o n of t h i s r a t i o by the number of m i l l i m o l e s of r a d i o -—6 a c t i v e 5-FU (4.09 x 10 m i l l i m o l e s ) g i v e s the t o t a l number of m i l l i m o l e s of 5-FU i n the 3.44 x 10~ 5 m i l l i m o l e s of tRNA. That -4 -5 i s , there was 4.2 x 10 m i l l i m o l e s of 5-FU i n 3.44 x 10 m i l l i m o l e s of tRNA. T h i s corresponds to an average of approx-imately 12 5-FU r e s i d u e s per tRNA molecule. The remaining f r a c t i o n s , corresponding to the tRNA i n F i g -ure 2.10, were pooled and 0.1 volumes of 0.1 molar magnesium c h l o r i d e was added followed by 2.5 volumes of 95% e t h a n o l p r e -cooled to -20 degrees c e n t i g r a d e . The p r e c i p i t a t e d tRNA con-s i s t e d of a mixture of FU-tRNA and components c o n t a i n i n g l i t -t l e or.no 5-FU. In order to improve 1 9F-nmr s i g n a l to noise i t was d e s i r a b l e to remove the u n f l u o r i n a t e d components. An en-richment procedure of t h i s type had been de v i s e d using DEAE-c e l l u l o s e chromatography (see s e c t i o n 2.4). P r e c y c l e d Whatman DE 32 (microgranular) was poured i n t o a 5 m i l l i l i t e r p i p e t which had a g l a s s wool stopper at the bottom end. The column was washed with the b u f f e r d e s c r i b e d i n s e c t i o n 2.4 c o n t a i n i n g 1 molar sodium c h l o r i d e . When the A260 r e a < ^ i n 9 was l e s s than 0.05 i t was e q u i l i b r a t e d with 3 volumes of 0.325 molar sodium c h l o r i d e i n the same b u f f e r . An apparatus s i m i l a r to the one shown in F i g u r e 2.6 was c o n s t r u c t e d to generate a concave upward sodium c h l o r i d e g r a d i e n t of i n c r e a s i n g c o n c e n t r a -t i o n . The mixing r e s e r v o i r had a diameter of 1.8 c e n t i m e t e r s . I t was connected to a standard 10 m i l l i l i t e r graduated c y c l i n d e r - 88 -Tube Number Figure 2.11. A DEAE-c e l l u l o s e s a l t g r a d i e n t of the tRNA f r a c t i o n a t e d in Fi g u r e 2.10. Each tube re p r e s e n t s 1.5 m i l l i -l i t e r s of e l u e n t . The l i n e connecting the s o l i d dots r e p r e -sents A260 values of the e l u e n t while the dotted l i n e con-n e c t i n g the hollow c i r c l e s i n d i c a t e s the D.P.M. values f o r 0.5 m i l l i l i t e r a l i q u o t s . The estimated s a l t c o n c e n t r a t i o n i s i n d i c a t e d by the l i n e connecting the t r i a n g l e s . - 89 -(1 centimeter diameter) v i a a g l a s s tube siphon. The mixing r e s e r v o i r was f i l l e d with approximately 20 m i l l i l i t e r s of the b u f f e r d e s c r i b e d i n s e c t i o n 2.4 and c o n t a i n i n g 0.325 molar s o d i -um c h l o r i d e . About 10 m i l l i l i t e r s of the same b u f f e r c o n t a i n i n g 0.6 molar sodium c h l o r i d e was added to the graduated c y c l i n d e r . A p o r t i o n of the tRNA p e l l e t obtained from the preceding e x p e r i -ment was d i s s o l v e d i n a minimal amount of the low s a l t b u f f e r and washed onto the e q u i l i b r a t e d D E A E - c e l l u l o s e column. Separa-t i o n of normal tRNA from FU-tRNA i s ev i d e n t from the e l u t i o n p r o f i l e and the corresponding D.P.M. measurements which are given i n F i g u r e 2.11. They were obtained as p r e v i o u s l y d e s c r i b e d f o r Fig u r e 2.10. E v a l u a t i o n o f the areas underneath the a n < ^ D.P.M. curves of Fi g u r e 2.11 i n d i c a t e that the enrichment technique was s u c c e s s f u l . The t o t a l r a d i o a c t i v i t y corresponds to 15,354 D.P.M. —6 or 6.92 x 10 m i l l i c u r i e s . With a s p e c i f i c a c t i v i t y of 10.26 m i l l i c u r i e s per m i l l i m o l e of r a d i o a c t i v e 5-FU t h i s corresponds -7 to 6.74 x 10 m i l l i m o l e s of r a d i o a c t i v i t y i n 0.11 m i l l i g r a m s (4.22 x 10~ 6 m i l l i m o l e s ) of tRNA. The t o t a l 5-FU i n c o r p o r a t i o n -5 i s then equal to 6.92 x 10 m i l l i m o l e s . T h i s corresponds to about 16 5-FU molecules per tRNA molecule. T h i s experiment provided evidence that 5-FU i n c o r p o r a t i o n i n t o the b a c t e r i a l tRNA was ex t e n s i v e (averaging about 12 5-FU per molecule of tRNA). I t a l s o demonstrated that enrichment of the FU-tRNA was e f f e c t i v e i n removing the n o n - f l u o r i n a t e d com-ponent and i n c r e a s i n g the average number of 5-FU re s i d u e s per molecule. - 90 -2.7 1 9F-FT nmr Spectroscopy of FU-5SrRNA F e a s i b i l i t y was i n i t i a l l y demonstrated with unfractionated FU-tRNA. Samples were prepared from E. c o l i B c e l l s grown according to the procedure given in section 2.2. The FU-tRNA was isolated as outlined in section 2.3 except that the tRNA fraction instead of the 5SrRNA fraction was collected and pre-c i p i t a t e d following Sephadex G-100 chromatography (see Figure 2.3). The f i n a l l y o p h i l i z e d product from step V of section 2.3 was dissolved in deuterium oxide (D2O) and r e - l y o p h i l i z e d . This was repeated a second time with to y i e l d unfractionated FU-tRNA having 15 &26O u n i t s P e r milligram per m i l l i l i t e r . The sample was then dissolved in a D 20 buffer of 0.01 molar sodium cacodylate, 0.002 molar EDTA (disodium salt) and adjusted to pH 7 with sodium deuteroxide. The f i n a l sample concentration was approximately 11 milligrams per m i l l i l i t e r . The FT '.^ F-nmr spectra of FU-tRNA, at 16 degrees centigrade and 72 degrees centigrade., are shown in figure 2.12. They were obtained on the departmental Varian XL 100 FT nmr spectrometer equipped with a Varian 620 L computer. Magnetic f i e l d strength was s t a b i l i z e d using an internal D 20 lock. The acquisition time was 0.2 seconds, with a s e n s i t i v i t y enhancement time constant of 0.1 seconds and a t o t a l time of 1.5 seconds between successive 90 degree pulses. The f i r s t FT 1 9F-nmr spectra of FU-5SrRNA were obtained on the same spectrometer mentioned above. Samples were prepared - 91 -6 (p.p.m.) i — i — i — i — i — i — i — i — i — i — r 8 6 4 2 0 Figure 2.12. FT 1 9F-nmr spectra of unfractionated native (16UC) and heat-denatured (76 C) 5-fluorouracil transfer-RNA. Total tRNA concentration i s 0.627 O.D. (260 nm)t or about 11 mg tRNA per ml, in a buffer of 0.01M cacodylate, 2mM EDTA (disodium s a l t ) , in with a pH meter reading of 7.55. Both spectra are plotted with 5 Hz/point, based on 4000 transients (16°C) or 1000 transients (76 C), with a s e n s i t i v i t y enhancement time constant of 0.1 sec. Chemical s h i f t , 6, in parts per m i l l i o n , i s referred to the chemical s h i f t for free 5-flu o r o u r a c i l at the same temperature in the same buffer. Heat denaturation of this sample was reversible. - 92 -from E. c o l i B c e l l s grown a c c o r d i n g t o the procedure g i v e n i n s e c t i o n 2.1. I s o l a t i o n and enr.ichment were a c c o r d i n g t o the methods o u t l i n e d i n s e c t i o n s 2.3 and 2.4. The e n r i c h e d FU-5SrRNA samples were l y o p h i l i z e d t w i c e a g a i n s t and then d i s s o l v e d i n a b u f f e r c o n s i s t i n g o f 0.02 molar sodium c a c o -d y l a t e (pH 7) c o n t a i n i n g 0.01 molar EDTA (dosodium s a l t ) and 0.02 molar magnesium c h l o r i d e . Sample c o n c e n t r a t i o n was ob-t a i n e d from A 2 6 0 m e a s u r e m e n t s where a 1 m i l l i g r a m per m i l l i -l i t e r s o l u t i o n o f FU-5SrRNA c o r r e s p o n d e d t o 21 A2gQ u n i t s ( 11). The 1 9F-nmr s p e c t r a o f FU-5SrRNA, a t 35 degrees c e n t i g r a d e and 72 degrees c e n t i g r a d e , are compared t o monomeric 5-FU and 5-f l u o r o - d e o x y - u r i d i n e monophosphate i n F i g u r e 2.13. When the sample a t 72 degrees c e n t i g r a d e was r e t u r n e d t o 35 degrees the spectrum was i d e n t i c a l t o the o r i g i n a l 35 degree spectrum p r i o r to heat d e n a t u r a t i o n . A spectrum was a l s o o b t a i n e d , a t 35 degrees c e n t i g r a d e , f o r an FU-5SrRNA sample which c o n t a i n e d no magnesium c h l o r i d e . T h i s i s shown i n F i g u r e 2.14. The 35 degrees 1 9F-nmr spectrum shown i n F i g u r e 2.13 i s d i v i d e d i n t o 4 r e g i o n s l a b e l e d a, b, c, and d. They a r e r e s p e c t i v e l y c e n -t e r e d about 6.6, 4.6, 3.6, and 2.1 p.p.m. r e l a t i v e t o 5-FU. The e n t i r e c h e m i c a l s h i f t range t h a t encompasses these 4 r e -g i o n s i s about 8 p.p.m.. The spectrum o f FU-5SrRNA i n b u f f e r c o n t a i n i n g no magnesium ( F i g u r e 2.14) i n d i c a t e s o n l y two p r o -minent peaks a t 1.9 and 4.9 p.p.m.. A s p i n - l a t t i c e r e l a x a t i o n time ( T ^ ) , a t 94.1 MHz, f o r the t h e r m a l l y d e n a t u r e d sample was o b t a i n e d from the s i g n a l t o - 93 -5F-deoxyUMP I 5 F U — i i 1 1 1 — 8 6 4 2 0 6 (p.p.m.) F i g u r e 2.13. FT 1 9F-nmr s p e c t r a of 5 - f l u o r o u r a c i l (5-FU) i n v a r i o u s molecules: (Top) Approximately equimolar mixture (each 0.0075 M) of FU and 5-fluoro-deoxy-UMP i n 0.01 M Cacodylate D 2 O b u f f e r , pH 7. This sample was used to determine c o r r e c t phase a d j u s t -ment for t h i s s p e c t r a l range. (Middle) 3 x 10~ 4 M FU-5SrRNA at 35°C, 30,000 t r a n s i e n t s , 0.01 M Cacodylate b u f f e r , pH meter reading 7.6 i n D 2 O . The four r e s o l v e d groups of peaks are l a b e l e d from a to d. (Bottom) 3 x 10~ 4 M FU-5SrRNA at 72°C, 10,000 t r a n s i e n t s , 0.01 M Cacodylate b u f f e r , pH meter reading 7.6 i n D 20. The dominant s i g n a l , l a b e l e d "b", corresponds to FU r e s i d u e s which are exposed to the e x t e r n a l s o l u t i o n (see t e x t ) . No proton d e c o u p l i n g was employed i n any of the s p e c t r a . The b u f f e r f o r the RNA sample a l s o contained 20 m i l l i m o l a r magnesium c h l o r i d e (13). - 94 -\ 1 1 1 4 1 + 1 f B 6 4 2 0 6(p.p.m.) F i g u r e 2.14. The FT 1 9F-nmr spectrum of FU-5SrRNA i n 0.02 molar sodium cacodylate D 20 b u f f e r c o n t a i n i n g 0.01 molar EDTA. The absence of monovalent (Na +) and d i v a l e n t (Mg + +) has caused a l -t e r a t i o n s i n the spectrum. The s p e c t r a l c o n d i t i o n s are the same as those given i n Fig u r e 2.13. - 95 -A A T + P D ( s e c o n d s ) F i g u r e 2.15. The T-, d e t e r m i n a t i o n o f t h e r m a l l y d e n a t u r e d FU-5SrRNA (see F i g u r e 2.13) employing a 90°, T , 90 p u l s e t e c h -n i q u e . A c o n s t a n t number of t r a n s i e n t s (1000) were a c q u i r e d f o r each d a t a p o i n t . The r e c i p r o c a l of the s l o p e o f B, e q u a l t o T]_, i s 0.6 seconds w i t h a s t a n d a r d d e v i a t i o n o f about 12%. AT and PD are a c q u i s i t i o n and p u l s e d e l a y times r e s p e c t i v e l y . - 96 -noise r a t i o of a constant number of transients (1000) at var-ious pulse delays and acquisition times. The data is indicated by the two graphs shown in Figure 2.15. A linear regression of the data points in Figure 2.15(b) gives a best f i t straight l i n e with slope -1.51 and standard deviation of 0.17. The rec i p r o c a l of the slope, equal to T-^ , i s 0.6 seconds with a standard deviation of about 12%. The 1 9F-nmr spectra of FU-5SrRNA, at 254 MHz, were ob-tained on an HX270 BrUker FT nmr spectrometer at the Univer-s i t y of Alberta Biochemistry Department in Edmonton. The assistance of Dr. Brian Sykes, whose e a r l i e r work on the f l u o r -ine labeled alkaline phosphatase enzyme led to formulation of this study (12-15), i s greatly appreciated. The samples were prepared as above and dissolved in either a D 20 or an H 20 phosphate buffer (pH 7) that contained 0.01 molar phosphate, 0.01 molar magnesium'chloride and 0.1 molar sodium chloride. The H 20 and D20 spectra are shown in Figures 2.16 and 2.17 respectively. Also included in each of these figures i s a spectrum in which a convolution difference technique has been employed to enhance the spectral resolution. The peak posi-tions for the D 20 and H 20 spectra are tabulated in p.p.m. rel a t i v e to 5-FU in Table 2.1. T^ estimates of individual peaks were obtained from the graphs shown in Figure 2.18. This data was acquired from the signal to noise r a t i o of peak i n t e n s i t i e s obtained during a 180°-T-90°-AT pulse sequence where x was varied between 0.03 and 0.6 seconds. The T-, - 97 -6 5 6 (ppm) 6 5 6 (ppm) F i g u r e 2.16. The FT 1 9F-nmr spectrum of FU-5SrRNA i n H 20 b u f f e r (see t e x t ) . T h i s spectrum was obtained at 254 MHz on a Bruker WH-270 nmr spectrometer. The top spectrum was obtained from the F o u r i e r t r a n s f o r m a t i o n of the f r e e i n d u c t i o n decay accumulated from 38,000 time-domain t r a n s i e n t s (5 mm sample). T y p i c a l ex-p e r i m e n t a l parameters were: 8k time-domain data s e t , 5000 Hz s p e c t r a l width, 0.5 sec r e l a x a t i o n d e l a y , quadrature d e t e c t i o n , 10 microsecond p u l s e width (approximately 9 0 ° ) , and s i g n a l to noise enhancement by e x p o n e n t i a l m u l t i p l i c a t i o n to give a l i n e broadening of 5 h e r t z . The bottom spectrum i s the c o n v o l u t i o n d i f f e r e n c e which improves the r e s o l u t i o n of i n d i v i d u a l peaks. 6 [m>m.) 5FdUMP H 1 1- 2 6 (ppm) F i g u r e 2.17. The FT 1 9F-nmr spectrum o f FU-5SrRNA i n the D 20 b u f f e r d e s c r i b e d i n the t e x t ( A ) . The s p e c t r o m e t e r and the s p e c t r a l parameters are the same as i n d i c a t e d i n F i g u r e 1.16. A c o n v o l u t i o n d i f f e r e n c e t e c h n i q u e was employed t o improve the s p e c t r a l r e s o l u t i o n o f the i n d i v i d u a l peaks ( B ) . Spectrum G g i v e s the r e l a t i v e p o s i t i o n s o f the 5 - f l u o r o - 2 ' - d e o x y u r i d i n e monophosphate (5FdUMP) and 5 - f l u o r o u r a c i l (5-FU). - 9 9 -Figure 2.18. The T^ d e t e r m i n a t i o n of i n d i v i d u a l peaks of FU-5SrRNA (in D 20 b u f f e r ) a t 254 MHz. A 180°, T, 90° pulse se-quence was employed to c o l l e c t 4 data p o i n t s f o r each peak. In t h i s f i g u r e s i g n a l - t o - n o i s e r a t i o s (from 1000 t r a n s i e n t s ) are p l o t t e d a g a i n s t time d e l a y , t , i n seconds. In order to determine T-^  v a l u e s the data p o i n t s were assumed to l i e along an e x p o n e n t i a l . - 100 -D 90 H„0 1 A = p.p.m. (D-O) p.p.m. p.p.m. c 2. Peak from 5-FU from 5-FU p.p.m. (HgO) 1 7.64 (?) 7.02 (?) 0.62 (?) 2 7.14 6 .70 0.44 3 6.52 6 .00 0.52 4 5.78 5.22 0.36 5 5 .06 4.90 0.36 6 4.91 4.56 0.35 7 4 .28 a) 4.00 a) 0 . 28 b) 3.76 b) 0.52 8 3.00 2.7 0.3 9 2.64 2.20 0.44 10 1.56 (?) 1.36 (?) 0.4 (?) Table 2.3. The peak p o s i t i o n s measured i n p.p.m. r e l a t i v e t o 5-FU f o r the H 20 and D 20 s p e c t r a of FU-5SrRNA o b t a i n e d a t 254 MHz. The 5-FU p o s i t i o n i n H2O i s s h i f t e d 0.2 p.p.m. d o w n f i e l d from the D 20 spectrum (14). T h i s c o r r e c t i o n i s i n c l u d e d i n the d e t e r m i n a t i o n o f the peak p o s i t i o n s o f the H 20 spectrum. v a l u e s were o b t a i n e d from the x - i n t e r c e p t where M = 0 and T, = fc0 . These v a l u e s a r e g i v e n i n Table 2.4. In 2 The f i n a l e xperiment o b t a i n e d on the Bruk e r HX270 i n v o l v e d a n u c l e a r Overhauser enhancement s t u d y . T h i s i s shown i n F i g u r e 2.19. Broad band i r r a d i a t i o n o f the p r o t o n s a t 270 MHz reduces the peak i n t e n s i t y t o zero -; e x c e p t f o r a. v e r y s l i g h t amount o f peak i n t e n s i t y as i n d i c a t e d . The presence o f monovalent c a t i o n s s t a b i l i z e the RNA s t r u c -t u r e by n e u t r a l i z i n g the cou l o m b i c r e p u l s i o n of the n e g a t i v e l y charged p hosphates. The r e s u l t i s a t i g h t e r and more e f f i c i e n t s t a c k i n g o f base p a i r s i n h e l i c a l r e g i o n s . T h i s i s e v i d e n t from - 101 -A ' 8 ' 6 ' i ' 2 ' 0 R R M . F i g u r e 2.19. A n u c l e a r Overhauser enhancement experiment of FU-5SrRNA (in D 20 b u f f e r ) at 254 MHz. The 1 9 F spectrum shown in A (from 1000 t r a n s i e n t s ) was obtained immediately f o l l o w i n g broadband s a t u r a t i n g i r r a d i a t i o n of the corresponding 'H-nmr spectrum. The decoupling o s c i l l a t o r was turned o f f d u r i n g 1 9 F data a c q u i s i t i o n . Spectrum B was obtained from 1000 t r a n s i e n t s f o r the same system, but i n the absence of i r r a d i a t i o n at the JH resonant f r e q u e n c i e s . - 102 -t "0 "1 -ln 2 2 0.3 0.43 3 0.27 0.40 4 0.23 0.33 5 0.27 0.39 6 0.23 0.33 7 0.20 0.29 8 0.24 0.35 9 0.24 0.35 Table 2.4. T^ de t e r m i n a t i o n s f o r the i n d i v i d u a l peaks of the D 20 sample of FU-5SrRNA obtained at 254 MHz. T, values were estimated from t ^ v a l u e s ( x - i n t e r c e p t where Mz=0) of F i g u r e 2.18 a l i n e a r dependence of the t r a n s i t i o n temperature on the l o g a -rithm of the a c t i v i t y of the monovalent c o u n t e r i o n s (16). The d i v a l e n t c a t i o n magnesium a l s o appears to be important i n s t a -b i l i z i n g RNA s t r u c t u r e . The 5SrRNA of E. c o l i has at l e a s t 4 strong magnesium b i n d i n g s i t e s (17). The 94 MHz spectrum of FU-5SrRNA was obtained i n a b u f f e r c o n t a i n i n g only the d i v a l e n t c a t i o n magnesium (at a c o n c e n t r a t i o n of about 20 magnesium per FU-5SrRNA m o l e c u l e ) . I t i s l i k e l y t h a t the molecules i n the 94 MHz sample e x i s t i n a somewhat l e s s compact s t a t e than the FU-5SrRNA sample used f o r the 234 MHz study where 0.1 molar NaCl was a l s o p r e s e n t . However, the o v e r a l l r i g i d i t y of the molecule i s not l i k e l y to be a l t e r e d s i g n i f i c a n t l y s i n c e the sedimentation c o e f f i c i e n t (S~» ), obtained by u l t r a - c e n t r i f u -v 20,w' 2 g a t i o n , i s f a i r l y i n s e n s i t i v e to changing c o u n t e r i o n concen-t r a t i o n (18 ) . - 103 -2.8 Laser Raman Spectroscopy of 5SrRNA and •; FU-5SrRNA 2.8.1 I n t r o d u c t i o n Raman spectroscopy has been used e x t e n s i v e l y to i n t e r p r e t c o n f o r m a t i o n a l p r o p e r t i e s of b i o l o g i c a l molecules. These i n -t e r p r e t a t i o n s are based l a r g e l y on observed changes i n the i n t e n s i t i e s of v a r i o u s Raman l i n e s due to changing macromole-c u l a r conformation. The major purpose of the experiments shown in t h i s s e c t i o n was to compare the conformations of normal 5SrRNA (N-5SrRNA) and FU-5SrRNA. The Raman s p e c t r a of an RNA molecule i s the sum of the r i n g v i b r a t i o n s of the bases which compose i t p l u s v i b r a t i o n a l c o n t r i b u t i o n s from the ribophos-phate backbone. Th i s s e c t i o n i s d i v i d e d i n t o two p a r t s . The f i r s t p a r t ( s e c t i o n 2.8.2) g i v e s Raman s p e c t r a of aqueous s o l u t i o n s of u r a c i l , 2-deoxyuridine, 2-deoxyuridine monophosphate, 5-FU, 5- f l u o r o - 2 - d e o x y u r i d i n e , and 5- f l u o r o - 2 - d e o x y u r i d i n e monophos-phate. Spectra of p o l y c r y s t a l l i n e u r a c i l and p o l y c r y s t a l l i n e 5-FU were a l s o o b t a i n e d . Comparison of a l l these s p e c t r a pro-vided i n f o r m a t i o n about the e f f e c t s of 5 - f l u o r o - s u b s t i t u t i o n upon the r i n g v i b r a t i o n s o f the u r a c i l base. I n t e n s i t y changes due to 5 - f l u o r o - s u b s t i t u t i o n i s a necessary c o n s i d e r a t i o n f o r the i n t e r p r e t a t i o n of the FU-5SrRNA s p e c t r a . In s e c t i o n 2.8.3 the Raman s p e c t r a of N-5SrRNA and FU-5SrRNA i n aqueous b u f f e r are g i v e n . Comparison of these s p e c t r a provided i n f o r m a t i o n about c o n f o r m a t i o n a l d i f f e r e n c e s and s i m i l a r i t i e s between the two molecules. - 104 -2.8.2 L a s e r Raman s p e c t r a o f 2 - d e o x y u r i d i n e 5 - f l u o r o - 2 - d e o x y u r i d i n e and r e l a t e d monomers Aqueous ( o r D 2 O ) s o l u t i o n s o f 50 m i l l i m o l a r 5-FU, 5 - f l u o r o -2 - d e o x y u r i d i n e , 5 - f l u o r o - 2 - d e o x y u r i d i n e m o n o p h o s p h a t e , o r 2-d e o x y u r i d i n e were p r e p a r e d w i t h d e i o n i z e d w a t e r c o n t a i n i n g 50 m i l l i m o l a r s o d i u m p e r c h l o r a t e . The s o d i u m p e r c h l o r a t e was u s e d a s an i n t e r n a l f r e q u e n c y and i n t e n s i t y s t a n d a r d . The a q u e o u s u r a c i l s a m p l e was o n l y 20 m i l l i m o l a r due t o i t s l o w e r s o l u b i l i t y and c o n t a i n e d no i n t e r n a l s t a n d a r d . The s o l u t i o n s were t i t r a t e d t o d e s i r e d pH v a l u e s w i t h s m a l l a l i q u o t s o f c o n -c e n t r a t e d s o d i u m h y d r o x i d e o r c o n c e n t r a t e d h y d r o c h l o r i c a c i d . A l l pH m e a s u r e m e n t s were o b t a i n e d w i t h a R a d i o m e t e r pH m e t e r 28, e q u i p p e d w i t h a R a d i o m e t e r t y p e GK 2322 C c o m b i n e d e l e c -t r o d e . Raman s a m p l e s were p r e p a r e d by l o a d i n g 20 m i c r o l i t e r a l i q u o t s o f s o l u t i o n a t t h e d e s i r e d pH i n t o Kimax m e l t i n g p o i n t c a p i l l a r y . t u b e s ( 0 . 8 - 1 . 1 mm i . d . ) . F o r t h e p o l y c r y s t a l -l i n e s p e c t r a o f u r a c i l and 5-FU t h e powder was a d d e d t o t h e same s i z e c a p i l l a r y t u b e s . Raman s p e c t r a o f t h e above s a m p l e s a r e p r e s e n t e d i n F i g -u r e s 2.20 - 2.24. A l l s p e c t r a were o b t a i n e d w i t h a Spex Ramalog 4 l a s e r Raman s p e c t r o m e t e r s y s t e m e q u i p p e d w i t h a S p e c t r a -P h y s i c s M o d e l 164 a r g o n i o n l a s e r (5145 a n g s t r o m s ) . Aqueous :(and D 2 O ) s a m p l e s were t r a n s v e r s e l y i l l u m i n a t e d w i t h 600 m i l -l i w a t t s o f l a s e r p o w e r . A s p e c t r a l s l i t w i d t h o f 7 - 9 cm ^ w i t h a s c a n s p e e d o f 0.2 wave numbers p e r s e c o n d and a p e r i o d o f 10 s e c o n d s was e m p l o y e d f o r a l l s o l u t i o n s p e c t r a . The - 105 -F igure 2.20. Laser Raman s p e c t r a of (a) p o l y c r y s t a l l i n e 5FU , (b) p o l y c r y s t a l l i n e U, and (c) 20 mM U ( n e u t r a l form) i n H-O. - 106 -co O) 00 o J I 1 I I I I I 1 1 1 1— 1 6 0 0 1 4 0 0 1 2 0 0 1 0 0 0 8 0 0 6 0 0 C M 1 Figure 2.21. Laser Raman spectra of 50 mM 5FU with 50 mM NaClO. (932 cm - 1 line) in H 20 at d i f f e r e n t pH. (a) pH 4.8 (b) pH 8 ( f i r s t ionization) (c) pH 11.3 (d) pH 12.1 (e) pH 13 (second ionization) - 107 -o CO CM CM CO i i I I I I I 1 1 1 1 — I L 1600 1400 1200 1000 800 600 C M " 1 F i g u r e 2.22. L a s e r Raman s p e c t r a o f n e u t r a l and a n i o n i c forms of dUrd and 5FdUrd i n H 20 c o n t a i n i n g 50 mM NaC10 4 (932 c m - 1 ) . (a) 50 mM dUrd, pH 5.6 ( n e u t r a l form) (b) 50 mM 5FdUrd, pH 4.8 ( n e u t r a l form) (c) 50 mM dUrd, pH 11 ( a n i o n i c form) (d) 50 mM 5FdUrd, pH 11 .(anionic form) - 108 -00 ' ' ' 1 I I -I 1 1— 1400 1200 1000 800 600 C M " Figure 2.23. Laser Raman s p e c t r a of n e u t r a l and a n i o n i c forms of dUrd and 5FdUrd i n D 20 c o n t a i n i n g 50 mM NaC10 4 (932 c m - 1 ) . (a) 50 mM dUrd, pH 6.2 ( n e u t r a l form) (b) 50 mM 5FdUrd, pH 5.8 ( n e u t r a l form) (c) 50 mM dUrd, pH 11.2 ( a n i o n i c form) (d) 50 mM 5FdUrd, pH 12.1 ( a n i o n i c form) - 109 -Fi g u r e 2.24. Laser Raman s p e c t r a of n e u t r a l and a n i o n i c forms of 5FdUrd and 5FdUMP i n H 20 c o n t a i n i n g 50 mM NaC10 4 (932 c m - 1 ) . (a) 47.5 mM 5FdUMP, pH 6 ( n e u t r a l form) (b) 50 mM 5FdUrd, pH 5.6 ( n e u t r a l form) (c) 47.5 mM 5FdUMP, pH 11.6 (ani o n i c form) (d) 50 mM 5FdUrd, pH 11 (ani o n i c form) - 110 -p o s i t i o n s of a l l Raman l i n e s (except aqueous U) were measured r e l a t i v e to the 932 wave number l i n e of the sodium p e r c h l o r a t e i n t e r n a l standard. S p e c t r a of p o l y c r y s t a l l i n e u r a c i l and 5-FU were obtained with 50 m i l l i w a t t s l a s e r power, s p e c t r a l s l i t width of approximately 6 wave numbers, scan speed of 2 wave numbers per second, and a p e r i o d of 2.5 seconds. U n c e r t a i n t i e s of ± 2 wave numbers are assigned to sharp peaks. I n t e r p r e t a -t i o n of these s p e c t r a are given i n Chapter 3. 2.8.3 Laser Raman s p e c t r a of normal 5SrRNA and FU-5SrRNA The RNA samples were prepared from normal and 5-FU t r e a t e d E:. c o l i B c e l l s a c c ording to the procedure o u t l i n e d i n s e c t i o n 2.3 using 2 x 190 centimeter Sephadex G-100 or G-75 columns. Samples from 5-FU t r e a t e d c e l l s were a l s o subjected to the procedure given i n s e c t i o n 2.4 to remove u n f l u o r i n a t e d 5SrRNA. D i a l y s i s of normal 5SrRNA samples was c a r r i e d out two times a g a i n s t 2 m i l l i m o l a r magnesium c h l o r i d e i n d e i o n i z e d water, once'against d e i o n i z e d water and then l y o p h i l i z e d . D i a l y s i s of the FU-5SrRNA sample d i d not produce r e s o l v a b l e s p e c t r a . Hence Raman s p e c t r a of FU-5SrRNA were obtained without d i a l y s i s a g a i n s t magnesium. Samples were prepared from f r e e z e - d r i e d normal or 5-FU c o n t a i n i n g 5SrRNA. The aqueous b u f f e r c o n s i s t e d of 0.01 molar phosphate adju s t e d to pH 7, 0.1 molar sodium c h l o r i d e , and 0.01 molar magnesium c h l o r i d e . Between 0.5 m i l l i g r a m s and 0.7 m i l l i g r a m s of normal or 5-FU c o n t a i n i n g 5SrRNA was - I l l -dissolved in 10 m i c r o l i t e r s of t h i s buffer. Using a 10 micro-l i t e r hypodermic syringe samples were placed in Kimax c a p i l l a r y tubes (size 0.8-1.10 millimeter i.d.) and centrifuged at 10,000 g for about 10 minutes. Samples were then transversally i l -luminated with a Spectra-Physics Model 164 argon ion laser (5145 angstroms). Spectra were obtained with a Spex Ramalog 4 laser Raman spectrometer system using a spectral s l i t width of 0.5 wave numbers. A scan speed of 0.5 wave numbers per second • 4 with a period of 10 seconds "' and a gain of 1 x 10 pc counts per second was employed. Raman spectra of the RNA samples are given in Figures 2.25 and 2.26. Figure 2.25 compares normal 5SrRNA before and after d i a l y s i s . In Figure 2.26 the Raman spectrum of normal 5SrRNA and -FU-5SrRNA are compared. The i n t e n s i t i e s of the Raman lines of polynucleotides are generally normalized to the 1100 wave number l i n e which i s due to the symmetric stretching mode of the PC^ groups that compose the ribophosphate backbone of these molecules. This l i n e i s independent of conformation provided ionic strength i s not d r a s t i c a l l y altered .(19). Hence normalization of the other Raman li n e s to the 1100 wave number l i n e provides a concentration independent measure of l i n e i n -t e n s i t i e s . In Table 2.3 the normalized i n t e n s i t i e s of the major li n e s shown in Figures 2.25 and 2.26 are given. The intensity values represent the average of at least 5 spectra and r e p r o d u c i b i l i t y of sharp well resolved l i n e s were within 5%. The baseline for the 1100 wave number l i n e was obtained - 112 -Figure 2.25. Raman s p e c t r a of aqueous samples of N-5SrRNA be-fore (B) and a f t e r d i a l y s i s (A). Methods f o r sample and b u f f e r p r e p a r a t i o n are o u t l i n e d i n s e c t i o n 2.8.3. - 113 -F i g u r e 2.26. Raman s p e c t r a o f a q u e o u s s a m p l e s o f N-5SrRNA (A) a f t e r d i a l y s i s and FU-5SrRNA (B). M e t h o d s f o r s a m p l e and b u f f e r p r e p a r a t i o n a r e o u t l i n e d i n s e c t i o n 2.8.3. - 114 -Frequency (cm-1) Or igin N-5SrRNA (before d i a l y s i s ) N-5SrRNA (after d i a l y s i s ) FU-5SrRNA 670 :• G 0.59 0.66 0.70 725 A 0.80 0.65 0.74 785 C,U 2.96 2.21 2.10 814 -OPO- ,1.65 1.65 1.73 110 0 P O 2 " 1.00 1.00 1.00 1242 U,C,A 1.96 1.17 1.16 1300 A,C 0.85 0.55 1321 G 1.53 1.31 1.36 1338 A 1.04 1.10 1485 A, G 1.89 2.21 2.06 1575 A, G 2.04 1.94 2.03 Table 2.5. Line i n t e n s i t i e s of the spectra shown in Figures 2.25 and 2.26. A l l i n t e n s i t i e s have been normalized to the 1100 wave number l i n e of the PO2"" group. by constructing a l i n e between points at 1120 and 1060 wave numbers on the Raman spectrum. The baseline for the 814, 785, 725 and 670 wave number l i n e s were made by joining the points at 840 and 650 wave numbers. Intensities of the Raman lines between 1210 and 1440 wave numbers were obtained by construc-tion of a baseline between these two points on a Raman spec-trum. F i n a l l y , for the li n e s at 1485 and 1575 wave numbers a baseline was constructed between the points at 1440 and 1800 wave numbers. The actual i n t e n s i t i e s of a l l these Raman lines were obtained by the construction of a v e r t i c a l l i n e from the peak maximum to i t s corresponding baseline and measuring the lengths of these l i n e s to the nearest 0.1 centimeters. - 115 -REFERENCES: CHAPTER 2 1. M. Demerec and E. Cahn, J . B a c t e r i o l . 65, 27 (1953). 2. T. S c h l e i c h and J . G o l d s t e i n , J . Moi. B i o l . 15, 136 (1966) . 3. In Sephadex, Gel F i l t r a t i o n i n Theory and P r a c t i c e , Appelbergs B o k t r y a k e r i , Uppsala, 1971; pages 31-49. 4. Reference 3; pages 24-26 and page 31. 5. I . I . K a i s e r , B i o c h e m i s t r y 8, 231 (1969). 6. I . I . K a i s e r , B i o c h e m i s t r y 9, 569 (1970). 7. In Whatman Laboratory Manual of Advanced Ion-Exchange  C e l l u l o s e s , Whatman L t d . , Maidstone; pages 8-9. 8. J . H i n d l e y , J . Moi. B i o l . 30, 125 (1967). 9. G.M. Rubin, J . B i o l . Chem. 248, 3860 (1973). 10. In Graduate Laboratory Manual Department of Biochemistry  F a c u l t y of Medicine, U.B.C., page 77. 11. M. L i t t , Bichem. Biophys. Res. Commun. 32, 506 (1968). 12. B.D. Sykes, H.I. Weingarten, and M.J. S c h l e s i n g e r , Proc. Nat. Acad. S c i . USA 71, 469 (1974). 13. W.E. H u l l and B.D. Sykes, J . Moi. B i o l . 98, 121 (1975). 14. W.E. H u l l and B.D. Sykes, B i o c h e m i s t r y 13, 3431 (1974). 15. W.E. H u l l and B.D. Sykes, B i o c h e m i s t r y 15, 1535 (1976). 16. V.A. B l o o m f i e l d , D.M. C r o t h e r s , and I. Tinoco i n P h y s i c a l  Chemistry of N u c l e i c A c i d s , Harper & Row, P u b l i s h e r s , New York, 1974; page 332. 17. B. Appel, V.A. Erdmann, J . S t u l z and T. Ackerman, Nuc.  A c i d . Res. 7, 1043 (1979). 18. H. Boedtker and G. K e l l i n g , Biochem. Biophys. Res. Comm. 29 , 758 (1967) . 1 9 M.C. Chen. R. Giege, R.C. Lord, and A. Rich, B i o c h e m i s t r y * 17, 3134 (1978) . - 116 -CHAPTER 3 FLUORINE NUCLEAR MAGNETIC RESONANCE SPECTROSCOPY OF E. COLI FU-5SrRNA While unpaired e l e c t r o n s ( u s u a l l y a r t i f i c i a l n i t r o x i d e ) s p i n - l a b e l s have been used f o r more than 10 years as probes of macromolecular s t r u c t u r e , the i n t r o d u c t i o n of a r t i f i c i a l nuc-l e a r s p i n - l a b e l s has so f a r been l i m i t e d to 1 3 C enrichment of s p e c i f i c carbons i n p r o t e i n s (1-2) and n u c l e i c a c i d s (3-4), and to f l u o r i n e - l a b e l i n g of a number of enzymes (5-7). T h i s work repre s e n t s the f i r s t 1 9F-nmr study of a f l u o r i n e l a b e l e d nucle-i c a c i d , FU-5SrRNA. The s p e c i f i c advantages of f l u o r i n e as a nuclear s p i n -l a b e l i n RNA are m a n i f o l d . F i r s t , of the four major bases only u r a c i l i s r e p l a c e d by 5-FU. T h i s means that only about 20 of the 120 bases i n 5SrRNA c o n t r i b u t e to the 1 9F-nmr spectrum. Therefore g r e a t e r s i m p l i f i c a t i o n i n s p e c t r a l assignment and a n a l y s i s i s o b t a i n e d . Second, the replacement of u r a c i l by 5-FU does not s i g n i f i c a n t l y a l t e r the f u n c t i o n of 5SrRNA (as judged by b i n d i n g to ribosomes) (8) or the tRNA (as judged by amino a c i d acceptor f u n c t i o n (9), o p t i c a l spectrum (9) or heat-d e n a t u r a t i o n p r o f i l e (10)) from E. c o l i . T h i r d , s i n c e 1 9F-nmr chemical s h i f t s are much more widely d i s p e r s e d than proton s h i f t s , one can expect to monitor r e l a t i v e l y s m a l l changes i n - 117 -the environments of v a r i o u s i n d i v i d u a l f l u o r o u r i d y l a t e s s i m u l -taneously (5). Fourth, compared to 1 3 C (even when n o n s e l e c t i v e l y (3) or s e l e c t i v e l y (4) e n r i c h e d or 1 5 N or 3 1 P nmr from n a t i v e tRNA (11-12), the f l u o r i n e l a b e l g i v e s much stronger nmr s i g -n a l s . The s p e c t r a shown i n Fig u r e 2.13, at 94.1 MHz were ob-ta i n e d from about 10,000 to 30,000 t r a n s i e n t s f o r FU-5SrRNA -4 c o n c e n t r a t i o n s of l e s s than 3 x 10 molar, while comparable 1 3 C or 3 1 P s p e c t r a would r e q u i r e of the order of 1,000,000 t r a n -s i e n t s u sing e i t h e r much higher RNA c o n c e n t r a t i o n or much l a r g e r sample volume. F i n a l l y , • t h e t 5 - F U l a b e l i s cheaper than com-m e r c i a l l y a v a i l a b l e 1 3 C - e n r i c h e d n u c l e i c a c i d bases by a f a c -tor of about 1000. The 94.1. MHz s p e c t r a of FU-5SrRNA, at 35 degrees c e n t i -grade and at 72 degrees c e n t i g r a d e , are presented i n Fig u r e 2.13 (13). Each of these s p e c t r a r e p r e s e n t s the s u p e r p o s i t i o n of the 1 9F-nmr s i g n a l s fromiabout 20 l a b e l e d bases. Neverthe-l e s s , at l e a s t four d i s t i n c t groups of peaks are r e s o l v e d at 35 degrees c e n t i g r a d e , and the heat-denatured s p e c i e s (72 degrees centigrade) i s s u f f i c i e n t l y unfolded such that v i r t u a l l y a l l the f l u o r i n e n u c l e i have a common chemical s h i f t (a sharp s i g n a l at about 5 p.p.m. downfield from f r e e 5-FU), which i m p l i e s a common chemical environment f o r most of the 5-FU r e s i d u e s i n denatured FU-5SrRNA. Moreover, the denatured spectrum i s cen-tered near the resonant frequency of free 5F-deoxyuridine mono-phosphate, i n d i c a t i n g t h a t t h i s chemical s h i f t corresponds to an environment i n which the f l u o r i n e - l a b l e i s exposed to - 118 -s o l u t i o n . Comparing the r e l a t i v e i n t e n s i t y o f the FU-5SrRNA f o r the 35 and 72 degrees s p e c t r a , a t t h i s f r e q u e n c y , i t i s p o s s i b l e t o e s t i m a t e t h a t a p p r o x i m a t e l y 25-35% o f the 5-FU r e s -i d u e s i n the n a t i v e FU-5SrRNA s t r u c t u r e are exposed t o s o l u t i o n . A 90°, T, 90° p u l s e sequence method was employed t o determine the s p i n - l a t t i c e r e l a x a t i o n time (T-^) f o r the heat d e n a t u r e d (72 degrees c e n t i g r a d e ) s p e c t r a . The method i s shown i n F i g u r e 2.15. From the d a t a a Tj. of 0.6 seconds was o b t a i n e d . F i n a l l y , the heat d e n a t u r a t i o n was r e v e r s i b l e s i n c e the low temperature spectrum appeared t o be i d e n t i c a l b e f o r e and a f t e r h e a t i n g t o 72 degrees c e n t i g r a d e . I n c r e a s e d magnetic f i e l d s t r e n g t h improved the r e s o l u t i o n o f the f o u r r e g i o n s which were d e s i g n a t e d t o the 94.1 MHz spec-trum shown i n F i g u r e 2.13. At 254 MHz ( F i g u r e 2.16 and 2.17) the "a" r e g i o n , which was o n l y a pronounced s h o u l d e r a t 94.1 MHz, c o n s i s t s o f a s l i g h t s h o u l d e r and t h r e e w e l l d e f i n e d peaks. The "b" r e g i o n , b e l i e v e d t o be due t o 5-FU r e s i d u e s exposed t o the s u r f a c e , appears as two c l o s e l y spaced narrow peaks. The "c" r e g i o n , a s h o u l d e r a t 94.1 MHz, i s a sharp w e l l d e f i n e d peak a t 254 MHz. F i n a l l y , the "d" r e g i o n appears as two peaks and a s l i g h t s h o u l d e r . Together the 254 MHz spectrum o f FU-5SrRNA c o n s i s t s o f e i g h t w e l l d e f i n e d peaks and two s h o u l d e r s ; a marked improvement i n r e s o l u t i o n over the 94.1 MHz spectrum. The peak p o s i t i o n s , i n p.p.m. r e l a t i v e t o 5-FU, are shown i n Table 2.3 f o r samples p r e p a r e d i n H 90 and D 90 b u f f e r . - 119 -In Table 2.1 the peak p o s i t i o n s f o r FU-5SrRNA i n D 20 and H 20 b u f f e r are g i v e n . For the spectrum of FU-5SrRNA i n D 20 b u f f e r the chemical s h i f t s are i n p.p.m. r e l a t i v e to 5-FU i n D 20. In H 20 5-FU i s s h i f t e d d ownfield by about 0.2 p.p.m. (15). The chemical s h i f t values of the H 20 sample of FU-5SrRNA are r e l a t i v e to t h i s new zero p o s i t i o n f o r 5-FU. The A term i n Table 2.3 i s the d i f f e r e n c e between these D 20 and H 20 chemical s h i f t s . The s m a l l e s t A valu e s are an i n d i c a t i o n of the g r e a t -e s t s o l v e n t e f f e c t s i n c e the 0.2 p.p.m. c o r r e c t i o n f o r the H 20 sample w i l l c a n c e l out s h i f t s o f exposed 5-FU r e s i d u e s which experience the g r e a t e s t s o l v e n t e f f e c t . Resonances 4, 5, 6, and 7a have the s m a l l e s t A v a l u e s ; the re g i o n a s s o c i a t e d with the peak p o s i t i o n of 5 f l u o r o - 2 ' - d e o x y u r i d i n e monophosphate. I n d i v i d u a l peaks were s u f f i c i e n t l y r e s o l v e d f o r an e s t i -mation of T-^  v a l u e s . A.180°, T, 90° pulse sequence method was employed where x was the d e l a y time f o l l o w i n g a 180° pulse (14). This pulse sequence i s i l l u s t r a t e d i n F i g u r e 3.1. The 180° pulse i n v e r t s the magnetization (M^) along the Z' a x i s of a coo r d i n a t e system which r o t a t e s at the Larmor p r e c e s s i o n f r e -quency. During a time d e l a y , x, the i n v e r t e d M z component w i l l decay by v a r i o u s r e l a x a t i o n processes i n an e f f o r t to r e t u r n to the e q u i l i b r i u m value (M Q)• R e l a x a t i o n of M z i s given by the f a m i l i a r Bloch equation: - 120 -(c) Figure 3.1. Determination of T-^  by 180 u, T, 90 u sequences, (a) M i s i n v e r t e d by a 180° pulse at time 0. (b) A f t e r a time x a 90 pulse r o t a t e s M to the y' (or -y') a x i s . (c) The i n i -t i a l amplitude of the FID a f t e r the 90° p u l s e , which i s propor-t i o n a l to the value of M at time x, i s p l o t t e d as a f u n c t i o n of x. Note that each p o i n t r e s u l t s from a separate 180°, x, 90° sequence. The p o i n t corresponding to (b) i s i n d i c a t e d by the arrow (14) . - 121 -Immediately a f t e r the p u l s e , at t=0, M Z ~ ~ M 0 - I n t e g r a t i o n of equation (1) g i v e s : M z = M o ( l - 2 e " t / T l ) (2). Equation (2) i m p l i e s that a p l o t of peak i n t e n s i t y ( s i g n a l to noise r a t i o ) versus x w i l l i n c r e a s e e x p o n e n t i a l l y from -M with i n c r e a s i n g x f o r a 180°, x, 90° pulse sequence. The p o i n t of i n t e r s e c t i o n with the x - i n t e r c e p t , where Mz=0 and t = t Q p r o v i d e s a ready e s t i m a t i o n of T^. T h i s i s given i n equation (3): t TT = N — „ = 1.4427t (3) . 1 l n 2 o P l o t s of i n t e n s i t y versus x of four d i f f e r e n t values of x were presented i n F i g u r e 2.18 f o r peaks 2-9. I t was assumed that the four data p o i n t s f o r each peak l i e along e x p o n e n t i a l curves and T-^  v a l u e s were determined a c c o r d i n g to the method d e s c r i b e d above. Estimated T^ v a l u e s f o r peaks 2-9 are given in Table 3.1. Peak Estimated T-L (seconds) 2 0.43 3 0.40 4 0.33 5 0 . 39 6 0.33 7 0.29 8 0.35 9 0.35 Table 3.1. The estimated T i values f o r the 254 MHz * 9F-nmr spectrum of FU-5SrRNA in D 20 b u f f e r . A 180 , x, '9.0 pulse sequence method was employed to o b t a i n these r e s u l t s . - 122 -The most important T^ r e l a x a t i o n mechanism for molecules in s o l u t i o n i s due to magnetic i n t e r a c t i o n s between f l u o r i n e and n e i g h b o r i n g n u c l e i which have magnetic d i p o l e s . In t h i s study f l u o r i n e r e l a x a t i o n i s expected to be l a r g e l y due to neighboring protons. As the RNA molecule tumbles the l o c a l magnetic f i e l d of the protons l o c a t e d near f l u o r i n e n u c l e i generate time dependent f l u c t u a t i o n s which i n t e r a c t with the f l u o r i n e ' s l o c a l magnetic f i e l d . Only f l u c t u a t i o n s which are at the n a t u r a l p r e c e s s i o n frequency of the f l u r o i n e nucleus w i l l cause r e l a x a t i o n . T h i s s o - c a l l e d d i p o l e - d i p o l e r e l a x a t i o n can be due to protons which are a p a r t of the 5-FU moiety ( i n -tramolecular) or protons from other p o r t i o n s of n u c l e i c a c i d which are proximal to the f l u o r i n e nucleus ( i n t e r m o l e c u l a r ) . I f t h i s r e l a x a t i o n i s e n t i r e l y i n t r a m o l e c u l a r then about 95% w i l l be caused by the n e a r e s t neighbor proton (15). There i s a negative s i x t h power dependence of d i p o l e - d i p o l e r e l a x a t i o n on the d i s t a n c e between c o n t r i b u t i n g n u c l e i . T h i s w i l l be shown s h o r t l y . Given the carbon-hydrogen bond l e n g t h and bond angle fo r a t y p i c a l v i n y l i c proton the d i s t a n c e between the 6-carbon proton and the f l u o r i n e atom has been estimated from the X-ray c r y s t a l s t r u c t u r e of 5-FU (16-17). T h i s i s shown to be 2.58 angstroms i n F i g u r e 3.2. H u l l e t . a l . (15) and D o d d r e l l e t . a l . (19) have expressed d i p o l e - d i p o l e r e l a x a t i o n times of a s p i n i ( 1 9 F ) r e l a x e d by spi n s j ( XH) i n terms of the f o l l o w i n g equations: - 123 -Figur e 3.2. The i n t r a m o l e c u l a r d i s t a n c e between f l u o r i n e and the n earest proton f o r 5 - f l u o r o u r a c i l . The carbon-carbon and c a r b o n - f l u o r i n e bond d i s t a n c e s and bond angles are based on the a v a i l a b l e X-ray c r y s t a l s t r u c t u r e s of 5-FU (16-17). For the v i n y l i c proton (bonded to Cg) the bond angle, C5=Cg—H, and bond d i s t a n c e , C5—H, were assumed to be the same as f o r ethy-lene (18). where, K = ( J j . ) y.2y.2fi2 = 5.0449 x 1 0 1 0 (angstroms) 6 ( 5 ) 1 u 1 j 2 (seconds) - 124 -s i n c e y^ = 2.5176 x 10 r a d i a n s / s e c o n d ' g a u s s y j = 2.6753 x 10 r a d i a n s / s e c o n d • g a u s s ,-27 (7) (8) and ti^ = 1.05443 x 10 e r g - s e c o n d s . (9) The f u n c t i o n s and F 2 i n t e r m s o f s p e c t r a l d e n s i t y f u n c t i o n s (J) a r e g i v e n by: and F x = J ( c o i - o ) j ) +• 3.1(0^) + 6J (tn^+di^) ? 2 =' F 1 + 4J(0) + 6J ( a)j) (10) (11) where to^ and oOj a r e t h e r e s o n a n c e f r e q u e n c i e s , i n r a d i a n s p e r s e c o n d , o f f l u o r i n e ( i ) and p r o t o n s ( j ) a t a g i v e n m a g n e t i c f i e l d s t r e n g t h . T h e s e v a l u e s a r e g i v e n i n T a b l e 3.2 f o r t h e t h r e e most commonly e m p l o y e d m a g n e t i c f i e l d s t r e n g t h s . The J F i e l d S t r e n g t h (KG) 23.5 63.4 84.6 1 9 p R e s o n a n c e F r e q u e n c y (MHz) 94 .1 254 . 0 338 .7 1 9 p R e s o n a n c e F r e q u e n c y , t0£.-- ( r a d i a n s / "second) 5.91 x 10 1.6 x 10 2.13 x 10 8 >H R e s o n a n c e F r e q u e n c y (MHz) 100 270 360 >H R e s o n a n c e F r e q u e n c y , ( ju j ( r a d i a n s / s e c o n d ) 6.28 x 10 8 1.7 x 10 9 2.26 x 10 9 T a b l e 3.2. The r e s o n a n c e f r e q u e n c y o f t h e p r o t o n and t h e f l u o r i n e n u c l e u s a t t h e t h r e e most commonly e m p l o y e d m a g n e t i c f i e l d s t r e n g t h s . t e r m s i n e q u a t i o n s 10 and 11 a r e s p e c t r a l d e n s i t y f u n c t i o n s f o r i s o t r o p i c m o t i o n and a r e o f t h e f o r m shown i n e q u a t i o n 12, T. J ( I U ) = c: 2 2 1+C0 T '*' c (12) - 125 -where x c i s the o v e r a l l m o l e c u l a r c o r r e l a t i o n t i m e . E q u a t i o n s 4 and 5 may be r e a r r a n g e d i n t o the f o l l o w i n g form: T i ' : ? r i j = x / K F i < 1 3 ) T 2 ? r i j = 2 / K F 2 ( 1 4 ) P l o t s o f e q u a t i o n s 13 and 14 f o r the resonance f r e q u e n c i e s o f f l u o r i n e and hydrogen, shown i n Table 3.2, are g i v e n i n F i g -u r e s 3.3-3.5. F i g u r e s 3.3-3.5 i n d i c a t e t h a t f l u o r i n e and T 2 v a l u e s are e q u a l f o r f a s t m o t i o n . However as the m o l e c u l a r t u m b l i n g r a t e slows down ( l a r g e r T ) T^ and T 2 d i v e r g e . The T 2 time con-t i n u e s t o d e c r e a s e w h i l e T^ reaches an a b s o l u t e minimum and then b e g i n s t o i n c r e a s e a g a i n . The minimum T^ v a l u e , c o r r e s -ponding T 2 v a l u e s , the T c a s s o c i a t e d w i t h d i v e r g e n c e o f T-^  and T 2, and x c a s s o c i a t e d w i t h a minimum T-^  are summarized f o r F i g -u r e s 3.3-3.5 i n Table 3.3. These v a l u e s assume t h a t r e l a x a t i o n i s due o n l y t o the 6-carbon p r o t o n o f the 5-FU base which i s 2.58 angstroms from f l u o r i n e . The e s t i m a t e d T^ v a l u e s f o r the i n d i v i d u a l f l u o r i n e peaks, a t 254 MHz, are g i v e n i n Table 3.1. The a c c u r a c y o f these r e -s u l t s were h i n d e r e d by the broadness o f the peaks and t h e i r r e l a t i v e l y c l o s e p r o x i m i t y t o each other.' T h e r e f o r e l a r g e un-c e r t a i n t i e s (about 30%) must be a s s o c i a t e d w i t h these v a l u e s . The t a b u l a t e d v a l u e s f o r T-, range between 0.29 and 0.43 seconds; CM Figure 3.3. Log p l o t s of T, x r and T 2 x r versus the log of the o v e r a l l molecular time (T ) at 94.1 MHz. These values are obtained from equations 13 and 14. The c a l c u l a t i o n s assume that the two r e l a x a t i o n times are e x c l u s i v e l y d i p o l a r and due only to the nearest neighbor proton l o c a t e d 2.58 angstroms (r) away from the f l u o r i n e . - 127 -- 128 -- 129 -F i e l d S trength (KG) T l (seconds) T2 (seconds) x at c minimum T-^  T l * T2 23.5 0.44 0 .11 2.5 x 10~ 8 T G > 5 x 10 -10 63.4 1.19 0.27 1 x 10~ 8 T c T > 2 .5 x 10 -10 84.6 1.59 0.34 7.9 x 10~ 9 > 1.5 x 10 -10 Table 3 .3 . Minimum T-^  v a l u e s , corresponding T 2 v a l u e s , the T a s s o c i a t e d with divergence of T->_ and T 2 , and T c a s s o c i a t e d with the minimum T-^  f o r F i g u r e s 3 . 2 - 3 . 4 . These values assume d i p o l a r r e l a x a t i o n v i a the 6-carbon proton of 5-FU. T^ and T 2 are p r o p o r t i o n a l to magnetic f i e l d s t r e n g t h . The x value a s s o c i a t e d with minimum T-^  and the divergence of T]_ cand T 2 are a l s o f i e l d dependent. s u b s t a n t i a l l y l e s s than the p r e d i c t e d minimum value of 1.19 seconds (see F i g u r e 3.3) due e x c l u s i v e l y to i n t r a m o l e c u l a r d i p o l e - d i p o l e r e l a x a t i o n v i a the proton n e a r e s t t o the f l u o r i n e n u c l e i of 5-FU. The s h o r t time s c a l e f o r these values im-p l i e s a r e l a t i v e l y r i g i d s o l u t i o n s t r u c t u r e f o r FU-5SrRNA. A x c value of 1 x 10 , which corresponds to a minimum T^ i s a reasonable value f o r the tumbling of a macromolecule the s i z e of 5SrRNA. The i n h e r e n t s t a b i l i t y of base p a i r i n g and base s t a c k i n g i n t e r a c t i o n s i n RNA molecules suggest that i n FU-5SrRNA the 5-BU bases are r i g i d l y s i t u a t e d and i n t e r n a l r o t a t i o n , which would i n c r e a s e both T^ and T 2 (15), i s h i g h l y improbable. T h i s i s v e r i f i e d by a nuc l e a r Overhauser enhancement (N.O.E.) e x p e r i -ment to be d i s c u s s e d s h o r t l y . Another source of T^ r e l a x a t i o n of f l u o r i n e i s chemical s h i f t a n i s o t r o p y . The presence of an a p p l i e d f i e l d causes the - 130 -e l e c t r o n i c c l o u d about the f l u o r i n e nucleus to c i r c u l a t e . Since f l u o r i n e i s not s p h e r i c a l l y symmetric ( l i k e the proton) a secondary magnetic f i e l d i s generated at the nucleus. As a molecule tumbles time dependent o s c i l l a t i n g components of t h i s f i e l d w i l l be generated which are p e r p e n d i c u l a r to the a p p l i e d f i e l d and c o n t r i b u t e to r e l a x a t i o n . H u l l and Sykes have con-cluded t h a t t h i s mechanism does not c o n t r i b u t e s i g n i f i c a n t l y to T-^  r e l a x a t i o n i n t h e i r study of the f l u o r o t y r o s i n e l a b e l e d a l k a l i n e phosphatase enzyme system at 94 MHz or 235 MHz (20). However they have shown that T 2 r e l a x a t i o n i s s i g n i f i c a n t l y -.. a f f e c t e d by f l u o r i n e ' s chemical s h i f t a n i s o t r o p y and t h i s e f -f e c t i n c r e a s e s i n p r o p o r t i o n to i n c r e a s e d magnetic f i e l d s t r e n g t h . Therefore at high magnetic f i e l d s t r e n g t h s p e c t r a l r e s o l u t i o n i s hindered d e s p i t e improved frequency s e p a r a t i o n of peaks s i n c e there i s a l s o a p r o p o r t i o n a t e i n c r e a s e i n l i n e width. The accurate measurement of T 2 r e l a x a t i o n times are not pos-s i b l e f o r the FU-5SrRNA s p e c t r a due to the e x t e n s i v e o v e r l a p -ping of peaks. In F i g u r e s 2.16 and 2.17 estimated l i n e widths, at h a l f amplitude, are i n the neighborhood of at l e a s t 200 h e r t z . Line widths a s s o c i a t e d with the s u r f a c e environment (peaks 5 and 6) appear to be somewhat narrower. I f the l i n e shapes of these peaks are assumed to be L o r e n t z i a n and l i n e widths at h a l f am-p l i t u d e equal to 2/T 2 rad sec (21) then the T 2 values of the i n d i v i d u a l peaks are a l l l e s s than 10 m i l l i s e c o n d s . - 131 -The above d i s c u s s i o n of T^ and T 2 r e l a x a t i o n times must be taken with c o n s i d e r a b l e r c a u t i o n . The 254 megahertz spectrum r e v e a l s o n l y 8 w e l l r e s o l v e d peaks. In E. c o l i 5SrRNA there are 20 u r a c i l s and our FU-5SrRNA i s expected to e x h i b i t at l e a s t 85% replacement (9). T h i s means that the i n d i v i d u a l peaks r e f e r r e d to i n the above d i s c u s s i o n are more a p p r o p r i a t e -l y c o n s i d e r e d as r e g i o n s . Each r e g i o n i s l i k e l y to be the s u p e r p o s i t i o n of more than one f l u o r i n e . Therefore l i n e widths and T-^  r e l a x a t i o n times of these regions are l i k e l y to be the net e f f e c t s of more than one f l u o r i n e . An N.O.E. experiment provided the most d e f i n i t i v e i n f o r -mation about the 5-FU l a b e l s i n FU-5SrRNA. The proton reson-ances were s a t u r a t e d with a strong 270 MHz frequency s i g n a l (the Larmor frequency of the proton) and the 1 9F-nmr spectrum obtained with a weaker r a d i o frequency s i g n a l at the Larmor frequency of f l u o r i n e (254 MHz). As shown i n Fig u r e 2.19 there i s almost complete l o s s o f the 1 9F-nmr spectrum under the con-d i t i o n of proton s a t u r a t i o n . T h i s i s expected f o r a molecule the s i z e of 5SrRNA assuming s p i n - l a t t i c e r e l a x a t i o n e x c l u s i v e l y due to a d i p o l e - d i p o l e mechanism (22) . In order to understand t h i s c o n c l u s i o n i t i s necessary to f i r s t c o n s i d e r r e l a x a t i o n of a two s p i n system (23). The energy l e v e l diagram f o r a system c o n s i s t i n g of two nuclear s p i n s , I and S, i s shown i n Fig u r e 3.6. The spin s can be a l i g n e d a g a i n s t the s t a t i c magnetic f i e l d , i n d i c a t e d by the a s u b s c r i p t , or i n the d i r e c t i o n of the s t a t i c f i e l d (the 3 - 132 -Fi g u r e 3 .6 . An energy l e v e l diagram of a system c o n s i s t i n g of two nuclear s p i n s , I and S. The s u b s c r i p t s , a and 3, r e f e r r e s p e c t i v e l y to alignment of the sp i n s opposed to and i n the d i r e c t i o n of the a p p l i e d magnetic f i e l d . The W terms represent r e l a t i v e t r a n s i t i o n p r o b a b i l i t i e s between s p e c i f i e d energy l e v e l s (see t e x t ) . - 1 3 3 -s u b s c r i p t ) , with the l a t t e r being the e n e r g e t i c a l l y more favor-able s i t u a t i o n . The four p o s s i b l e t r a n s i t i o n p r o b a b i l i t i e s needed to d e s c r i b e T^ r e l a x a t i o n during an N.O.E. experiment S I are i n d i c a t e d i n F i g u r e 3 . 6 . W-^  and represent r e s p e c t i v e l y the r a t e s f o r s i n g l e quantum t r a n s i t i o n s of an S s p i n , given that I remains unchanged, and f o r an I s p i n with the S s p i n re-maining unchainged. The s u p e r s c r i p t i n d i c a t e s the s p i n under-going the quantum t r a n s i t i o n . The t r a n s i t i o n p r o b a b i l i t y , WQ , i s f o r both s p i n s I and S undergoing t r a n s i t i o n s of the type IJ.SX—>InS or I „ S » I S „ . L a s t l y , W 0 i s the l i k e l i h o o d t h a t a 3 B a 3 a a 3 2 two s p i n s a l i g n e d i n the same d i r e c t i o n w i l l r e l a x at the same time. That i s , t r a n s i t i o n s of the type I S > I N S N or J r a a 3 3 I 0 S ; >I S . Noggle and Schirmer have shown that f o r a two 3 3 a a ^ s p i n system where S = I = h the f r a c t i o n a l enhancement of the i n t e g r a t e d i n t e n s i t y of the I s p i n , given that the S s p i n i s s a t u r a t e d , i s d e s c r i b e d by equation 1 5 ( 2 4 ) . w2 - w f T ( S ) = = ( 1 5 ) 2W| + W Q + W 2 The numerator, W 2 - WQ i s c a l l e d the c r o s s r e l a x a t i o n term. I t i s t r a n s i t i o n s a s s o c i a t e d with W 2 and WQ that cause the N.O.E. e f f e c t . The e f f e c t of s a t u r a t i n g the S s p i n s on the energy l e v e l diagram shown i n F i g u r e 3.5 i s to e q u a l i z e the s p i n popu-l a t i o n s of l e v e l s 1 and 2 as w e l l as 3 and 4. T h i s r e s u l t s i n a change i n the p o p u l a t i o n s of a l l the energy l e v e l s from t h e i r e q u i l i b r i u m values p r i o r to s a t u r a t i o n . Consequently the system - -134 -w i l l attempt to r e - e s t a b l i s h the e q u i l i b r i u m p o p u l a t i o n s i n S I these l e v e l s . N e i t h e r W^ or W-^  t r a n s i t i o n s cause net changes i n the p o p u l a t i o n s of these l e v e l s . However, W 2 attempts to r e - e s t a b l i s h the e q u i l i b r i u m p o p u l a t i o n by i n c r e a s i n g the popu-l a t i o n of energy l e v e l 4 through a corresponding decrease i n energy l e v e l 1. T h i s process w i l l f a c i l i t a t e an i n c r e a s e i n the a b s o r p t i o n i n t e n s i t y of the I s p i n s . W Q w i l l r e d i s t r i b u t e the p o p u l a t i o n s between energy l e v e l s 2 and 3 which at e q u i l -i b rium are e q u a l . I t e f f e c t s a decrease i n the p o p u l a t i o n of energy l e v e l 3 and an i n c r e a s e i n the p o p u l a t i o n of energy l e v e l 2. I t s net e f f e c t i s to decrease the i n t e n s i t y of the nmr r e -sonance of the I s p i n s . Hence WQ and W 2 t r a n s i t i o n s have oppos-ing e f f e c t s under the c o n d i t i o n of s a t u r a t i o n of one of the s p i n s . Solomon has d e r i v e d e x p r e s s i o n s f o r the t r a n s i t i o n proba-b i l i t i e s a s s o c i a t e d with F i g u r e 3.6 assuming that the resonance f r e q u e n c i e s of the two s p i n s , t o ' j and c o s are not equal (25). They are given by equations 16-19. , 2 2 2 h Y T Y S T c T c W Q = g— = — ; — : — (16) 10b 1 + ( t O j - C O g ) T c 1 + ( W j - C O g ) T e 2 2 2 ! 3 h T l Y S T c T c W,. = o = K 1 " , n. 6 , ^ 2 2 "2 ... 2 2 (17) 20b 1 + L 0 J T C 1 + W j T c - . 2 2 2 3h Y L Y S T C T C W l - _ , 6 , . 2 2 " K2 — ' T T ( 1 8 ) 20b 1 + a ) g T r 1 + W g T c - 135 -o v 2 2 2 W „ =: -£ = E (19) ^ 5b b 1 + ( C O j + W g ) 1 + ( c o j + c o g ) TC For a two s p i n system of 1 9 F (I) and XH (S) n u c l e i i n 5-FU y F and y H are t h e i r gyromagnetic r a t i o s . They are, r e s p e c t i v e -l y , 25,179 radians/seconds-gauss and 26,753 radians/seconds* gauss. The o)j and cos terms are equal to 2J[ • 254 x 10^ c y c l e s / second and 2j[ • 270 x 10 c y c l e s / s e c o n d r e s p e c t i v e l y . The b e x p r e s s i o n i s the i n t e r n u c l e a r d i s t a n c e between f l u o r i n e and the n e a r e s t neighbor proton (2.58 angstroms) of 5-FU. The molecules c o r r e l a t i o n time i s T c and ti' i s Planck's constant (in u n i t s of ergs per second) d i v i d e d by 2J[. Given the above para-meters K-^ , K 2, and can be computed. They are: K 1 = 1.6331 x 1 0 8 / s e c o n d s 2 K 2 = 2.4497 x 1 0 8 / s e c o n d s 2 8 2 K 3 = 9.7987 x 10 /seconds As shown by equations 16-19 t r a n s i t i o n p r o b a b i l i t i e s are governed by the molecular c o r r e l a t i o n time, T . In Figure 3.7 these t r a n s i t i o n p r o b a b i l i t i e s are p l o t t e d a g a i n s t T . For slow r o t a t i o n (T > 7.1 x 10 ^ ) WQ > W 2 and according to equa-t i o n 15 the f r a c t i o n a l enhancement, f-j. (S), i s then n e g a t i v e . That i s , s i n c e WQ t r a n s i t i o n s dominate fo r slow molecular motion the nmr spectrum due to I ( l 9 F ) w i l l decrease with s a t u r a t i o n of the S ( !H) s p i n s . For f a s t tumbling (t < 7.1 x 10 1 0 sec-onds) W~ > W„ and t r a n s i t i o n s of the type I-.,S >I„S_ w i l l 2 0 J j r a a / B B - 136 -Figure 3.7. Log-log plot of t r a n s i t i o n rates versus rotational correlation time for the system of Figure 3.6, in which I is F-5 and S is H-6 of 5 - f l u o r o u r a c i l . Fluorine relaxation i s taken as pure dipolar between F-5 and H-6, resulting from isotropic ro-tati o n a l d i f f u s i o n . - 137 -dominate. T h i s w i l l r e s u l t i n p o s i t i v e enhancement; an i n crease i n the s i g n a l due to the I s p i n s when the S s p i n s are s a t u r a t e d . S u b s t i t u t i o n of equations 16-19 i n t o equation 15 p r o v i d e s the f o l l o w i n g e x p r e s s i o n f o r the f r a c t i o n a l N.O.E. of u n l i k e s p i n s (5): area (with  l E i r r a d i a t i o n ) - area (without *H i r r a d i a t i o n ) area (without rH i r r a d i a t i o n ) 2 2 2 2 2 2 H 5 + 5c0j T + 6(t0j-C0g) T - (oOj+COg) X + Y F 10 + 7CO-|-^T^ + 4 (to j + t O g ) ^ + 9 (Uj~iiis)^'+ 6 U J 2 ( t O j - G O g ) ^.T4 2 2 4 2 2 4 6 U ) T (lO T-U) C) T _ OJ T (tO-r+tOc;) X 2 2 4 2 2 4 K ' + COj < (lOj+ C O g ) X + 3(L0-j--C0g) ( ( j O j + C O g ) X The graph of equation 20 as a f u n c t i o n of c o r r e l a t i o n time i s shown i n F i g u r e 3.8 assuming an 1 9 F resonance frequency of 254 megahertz and proton s a t u r a t i o n with 270 megahertz. When x c > — 8 1 x 10 (slow molecular r o t a t i o n ) then ^ ( S ) = -1, which c o r -responds to a complete l o s s of the l 9 F s i g n a l i n t e n s i t y . The observed c o l l a p s e of the FT 1 9F-nmr spectrum shown i n F i g u r e 2.19 when the protons are i r r a d i a t e d at t h e i r resonance frequency confirms a f r a c t i o n a l N.O.E. value f o r a l l the 1 9 F resonances of f-j-(S) = -1. T h i s implies' that c r o s s r e l a x a t i o n v i a WQ dominates and that the FU l a b e l s are slowly tumbling at —8 a c o r r e l a t i o n time given by x c > 1 x 10 sec. T h i s circumstance would be r e q u i r e d i f the 5FU l a b e l s were r i g i d l y s i t u a t e d (im-mobile) i n the 5FU-5SrRNA molecule and tumbling at i t s c o r r e l a -t i o n time. - 138 -Figure 3.8. F l u o r i n e - p r o t o n f r a c t i o n a l n u c l e a r Overhauser en-hancement f a c t o r , f j (S) , versus r o t a t i o n a l c o r r e l a t i o n time (log scale) for 5 - f l u o r o u r a c i l , computed from the t r a n s i t i o n r a t e s of F i g u r e 3.7. - 139 -I f FU-5SrRNA were a r i g i d sphere, i t s x r o t c a l c u l a t e d from the S t o k e s - E i n s t e i n equation (26), 4IIhR3 m  T r o t 3kT K ' in which h i s v i s c o s i t y , k i s Boltzmann's const a n t , T i s abso-l u t e temperature, and R i s the macromolecular r a d i u s (computed from a molecular weight of 40,000), would be about 10 nsec, assuming no water of h y d r a t i o n . A r i g i d FU-5SrRNA should t h e r e -f o r e e x h i b i t f-j-(S) = -1 as shown i n F i g u r e 3.8. T h i s c o r r e s -ponds to complete n u l l i n g of the 1 9 F s i g n a l on i r r a d i a t i o n of the H-6 proton. According to F i g u r e 3.8 there i s a marked v a r i -a t i o n of f-j-(S) with x r o t i n t h i s motional r e g i o n . T h i s means that even a s m a l l i n c r e a s e i n l o c a l f l e x i b i l i t y at the l a b e l e d s i t e should lead to a l a r g e i n c r e a s e i n f-j-(S) and incomplete n u l l i n g (or even an i n c r e a s e ) of the l 9 F peak on proton i r r a d i -a t i o n . Thus, the i n s t r u m e n t a l and molecular parameters of the FU-5SrRNA Overhauser experiment happen to f a l l i n a range such t h a t the N.O.E. experiment i s o p t i m a l l y tuned to d e t e c t l o c a l f l e x i b i l i t y at the l a b e l e d u r a c i l s of FU-5SrRNA. In c o n c l u s i o n , the 254 MHz 1 9F-nmr s p e c t r a of FU-5SrRNA (Figures 2.16 and 2.17) i n d i c a t e at l e a s t 10 d i f f e r e n t chemical s h i f t s which corresponds to 10 d i s t i n c t chemical environments. T h i s i s an improvement i n r e s o l u t i o n when compared to the 94.1 MHz spectrum (Figure 2.13) where only 4 groups of peaks are observed. In F i g u r e s 2.16 and 2.17 peaks 5 and 6 have the same chemical s h i f t as the s i n g l e peak obtained on heat d e n a t u r a t i o n ; - 140 -thus, t h i s p a r t of the spectrum probably corresponds to FU r e s -idues exposed to s o l u t i o n . In support of t h i s view, i t may be noted t h a t peaks 5 and 6 are much narrower than peaks s h i f t e d to e i t h e r s i d e , and t h e r e f o r e correspond to r e s i d u e s t h a t are more r o t a t i o n a l l y l a b i l e than the s h i f t e d and presumably " b u r i e d " peaks. Furthermore, T-^  measurements i n d i c a t e t h a t v i r t u a l l y a l l the observed peaks have r e l a t i v e l y s h o r t ( 0 . 3 - 0 . 4 seconds), i n agreement with the minimum c a l c u l a t e d t o occur f o r t" r 0£ = 10 nsec which corresponds to a r i g i d s o l u t i o n s t r u c t u r e . The 1 9 F - X H N.O.E. experimental r e s u l t s (Figure 2.19) are p a r t i c u l a r -l y s t r i k i n g . I t i s c l e a r that v i r t u a l l y the e n t i r e 1 9 F spectrum i s c ompletely n u l l e d on 1H i r r a d i a t i o n , d e f i n i t i v e l y c o n f i r m i n g (see F i g u r e 3.8) t h a t e s s e n t i a l l y a l l the f l u o r i n a t e d u r a c i l r e s i d u e s have r o t a t i o n a l c o r r e l a t i o n times approaching 10 nsec or l o n g e r . Furthermore, demonstration of the f u l l Overhauser n u l l i n g of the 1 9 F resonances (f-j-(S) = -1) confirms that T^-r e l a x a t i o n i s pure d i p o l a r , as assumed i n the t h e o r e t i c a l c a l -c u l a t i o n s (5). [ ^ S t r i c t l y speaking, the f-j-(S) = -1 r e s u l t can a l s o occur i n the presence of very r a p i d i n t e r n a l motion (5). However, such r a p i d motion would have the a d d i t i o n a l e f f e c t of narrowing the 1 9 F resonances to l i n e widths, l e s s than are ob-served. Thus, we may s a f e l y conclude that the T R O ^ . v a l u e s are in f a c t v ery long (10 nsec or g r e a t e r ) rather than very short._\ - 141 -REFERENCES: CHAPTER 3 1. M.W. H a n k a p i l l e r , S.H. Smallcombe, and J.D. R i c h a r d s , B i o c h e m i s t r y 12, 4732 (1973). 2. I.M. C h a i k e n , M.H. Freedman, J.R. L y e r l a , and J . S . Cohen, J . B i o l . Chem. 248, 884 (1973). 3. P.F. A g r i s , F.G. Fuj.iwara, C F . S c h m i d t , and R.N. Loeppky, N u c l e i c A c i d s Res. 2, 1503 (19 7 5 ) . 4. W.D. H a m i l l , D.M. G r a n t , W.J. H o r t o n , R. L u n d q u i s t , and S. Dickman, J . Am. Chem. Soc. 98, 1276 (19 7 6 ) . 5. W.E. H u l l and B.D. S y k e s , J . M o i . B i o l . 98, 121 ( 1 9 7 5 ) . 6. J . Bode, M. B l u m e n s t e i n , and M.A. F a f t e r y , B i o c h e m i s t r y 14, 1153 ( 1 9 7 5 ) . 7. D.T. Browne and J.D. O t v o s , B i o c h e m . B i o p h y s . Res. Comm. 6 8 , 907 (19 7 6 ) . 8. J . L . J o h n s o n , K.R. Yamamoto, P.O. W e i s l o g e l , and J . L . H o r o w i t z , B i o c h e m i s t r y 8, 1901 (1969) . 9. I . K a i s e r , B i o c h e m i s t r y 9, 569 ( 1 9 6 9 ) . 10. J . H o r o w i t z , C.-N. Ou, M. I s h a q , J . O f e n g a n d , and' J . B i e r -baum, J . M o i . B i o l . 88, 301 (19 7 4 ) . 11. D. G u s t , R.B. Moon, and J.D. R o b e r t s , P r o c . N a t . A c a d .  S c i . , U.S.A. 72, 4696 (1 9 7 5 ) . 12. M. Gueron and R.G. Shulman, P r o c . N a t . A c a d . S c i . , U.S.A. 12, 3482 ( 1 9 7 5 ) . 13. A.G. M a r s h a l l and J . L . S m i t h , J . Am. Chem.' S o c . 99 , 635 (1977) . 14. T . C F a r r a r and E.D. B e c k e r , i n P u l s e and F o u r i e r T r a n s - form NMR, A c a d e m i c P r e s s , New Y o r k , 1971; pages 20-22. 15. W.E. H u l l and B.D. S y k e s , B i o c h e m i s t r y 13, 3431 (1974). 16. L. F a l l o n , A c t a . C r y s t . B59, 2549 (1 9 7 3 ) . 17. DV-Veo.t and A. R i c h , J . Am. Chem. Soc. 91, 3069 (1 9 6 9 ) . - 142 -18. N. A l l i n g e r , i n Organic Chemistry, Worth P u b l i s h e r s , New York, 1971; page 133. 19. D. D o d d r e l l , V. Glushko, and A. A l l e r h a n d , J . Chem. Phys. 56, 3683 (1972). 20. W.E. H u l l and B.D. Sykes, J . Moi. B i o l . 98, 121 (1975). 21. A. C a r r i n g t o n and A.D. McLachlan, i n I n t r o d u c t i o n to  Magnetic Resonance, Harper & Row, New York, 1967; pages 9-10. 22. P. Balaram, A. Bothner-By, and J . Dadok, J . Am. Chem. Soc. 94, 4015 (1972). 23. J.H. Noggle and R.E. Schirmer, i n The Nuclear Overhauser  E f f e c t , Academic Press, New York, 1971. 24. Reference 23; page 16. 25. I. Solomon, Phys. Rev. 99, 559 (1955). 26. A.G. M a r s h a l l , i n B i o p h y s i c a l Chem., Wiley, New York, 1971; page 719. - 143 -CHAPTER .4 LASER RAMAN SPECTROSCOPY OF N-5SrRNA AND FU-5SrRNA 4 .1 Introduction Molecules are dynamic e n t i t i e s which undergo continuous nuclear and electronic motion. Besides o v e r a l l t r a n s l a t i o n a l motion, the nuclear movement can be described in terms of three s p e c i f i c types of motion; rotation of the molecule as a whole, molecular vibrations between chemically bonded nuclei in the molecule, and the spins of i n d i v i d u a l n u c l e i . Associated with the electrons are o r b i t a l motion about the nucleus and spin. The r o t a t i o n a l and v i b r a t i o n a l components of nuclear motion and the o r b i t a l motion of the electrons are described in quantum mechanical terms as energy eigenvalues which together represent the t o t a l internal energy of a given molecule. In the l a s t chapter nuclear spin and molecular rotation were considered. Raman spectroscopy provides a method for obtaining v i b r a t i o n a l energies (and in some cases rotational energies) of chemically bonded nuclei in molecules. Rigorous t h e o r e t i c a l interpretation of Raman spectra i s only applicable for small molecules of high symmetry, where group theory can be employed to determine the number and the kinds of energy leve l s associated with the molecules' normal v i b r a t i o n a l modes. Most molecules of b i o l o g i c a l interest are - 144 -l a r g e and asymmetric. A 5SrRNA m o l e c u l e c o n s i s t s o f 3,987 atoms which c o r r e s p o n d s t o 11,955 (3N-6) v i b r a t i o n a l degrees of freedom. Even the r i g o r o u s i n t e r p r e t a t i o n o f the r i n g v i -b r a t i o n s o f the p u r i n e s and p y r i m i d i n e s i s u n f e a s i b l e . S t i l l , much u s e f u l i n f o r m a t i o n can be o t b a i n e d by employing an e m p i r i -c a l approach. S t r e t c h i n g and bending modes of s p e c i f i c mole-c u l a r g r o u p i n g s occur a t c h a r a c t e r i s t i c f r e q u e n c i e s . In RNA m o l e c u l e s these s o - c a l l e d "group f r e q u e n c i e s " are due t o the c h a r a c t e r i s t i c r i n g v i b r a t i o n s o f the p u r i n e and p y r i m i d i n e bases and the r i b o p h o s p h a t e backbone. They are o u t l i n e d i n Table 2.3. The i n t e n s i t i e s o f many of these Raman l i n e s are a f f e c t e d by RNA c o n f o r m a t i o n . These e m p i r i c a l o b s e r v a t i o n s are based on o b s e r v e d changes i n Raman i n t e n s i t i e s of model com-pounds of p u r i n e and p y r i m i d i n e p o l y r i b o n u c l e o t i d e s (1) and tRNA i n both n a t i v e and d e n a t u r e d forms ( 2 - 4 ) . T h i s c h a p t e r p r e s e n t s Raman s p e c t r a o f N-5SrRNA and FU-5SrRNA ( S e c t i o n 4.3). The comparison o f the i n t e n s i t i e s o f v a r i o u s Raman l i n e s was used t o e v a l u a t e the e f f e c t s o f 5-FU i n c o r p o r a t i o n on 5SrRNA c o n f o r m a t i o n . A n e c e s s a r y c o n s i d e r a -t i o n o f t h i s s t u d y was the e f f e c t o f 5 - f l u o r o - s u b s t i t u t i o n on the r i n g v i b r a t i o n s o f the u r a c i l base s i n c e a l t e r a t i o n s i n the i n t e n s i t y o f the r i n g v i b r a t i o n s o f t h i s base c o u l d be m i s i n -t e r p r e t e d as changes i n FU-5SrRNA c o n f o r m a t i o n as compared t o N-5SrRNA. T h i s l a t t e r a s p e c t i s c o n s i d e r e d i n s e c t i o n i 4 . 2 . - 145 -4.2 L a s e r Raman Study o f 5-FU, 5 - F l u o r o - 2 ' - d e o x y u r i d i n e , and  5 - F l u o r o - 2 ' - d e o x y u r i d i n e Monophosphate To d a t e , X-ray ( 5 , 6 ) , 1 3C-nmr ( 7 ) , 1 9F-nmr ( 8 ) , and i n f r a -r e d s p e c t r o s c o p y (9,10) have been used t o examine the e f f e c t s of f l u o r i n a t i o n on the s t r u c t u r a l and c h e m i c a l p r o p e r t i e s o f the u r a c i l base. In o r d e r t o e v a l u a t e the e f f e c t o f 5 - f l u o r o -s u b s t i t u t i o n on the v i b r a t i o n a l p r o p e r t i e s o f the u r a c i l b ases, l a s e r Raman s p e c t r a o f p o l y c r y s t a l l i n e U and 5-FU, 5-FU i n ^ 0 , 5-f l u o r o - 2 -deoxy.urr i d i n e (5-FdUrd) i n H^O and D2O, and 5 - f l u o r o -2 1 - d e o x y u r i d i n e monophosphate (5-FdUMP) i n I^O were o b t a i n e d f o r comparison t o normal 2 ' - d e o x y u r i d i n e (dUrd) i n H^O and D2O. A g e n e r a l i z e d s t r u c t u r e f o r U, dUrd, 5-FU, 5-FdUrd, and 5-FdUMP i s shown i n F i g u r e 4.1. U and 5-FU each p o s s e s s d i s -s o c i a b l e p r o t o n s a t N - l and N-3. 4.2.1 U r a c i l and 5 - F l u o r o u r a c i l L a s e r Raman s p e c t r a o f p o l y c r y s t a l l i n e (powder) samples o f 5-FU and U were p r e s e n t e d i n F i g u r e s 2.20a and 2.20b. Peaks a s s o c i a t e d w i t h C-N s t r e t c h i n g v i b r a t i o n s (1226 cm f o r 5-FU; 1234 cm ^ f o r U) , and w i t h r i n g b r e a t h i n g motions (770 cm f o r 5-FU; 790 cm 1 f o r U) are s i m i l a r i n appearance f o r both compounds. The c a r b o n y l s t r e t c h i n g r e g i o n (1600-1700 cm "*") i s d i f f e r e n t f o r 5-FU than f o r U; the d i f f e r e n c e s are more p r o -nounced and more e a s i l y i n t e r p r e t e d f o r the c o r r e s p o n d i n g nuc-l e o s i d e s d i s c u s s e d i n s e c t i o n 4.2.2. - 146 -H X = H; R = H Uracil X = H; R = 2 1-deoxyribose dUrd X = F; R = H 5FU X = F; R = 2'-deoxyribose FdUrd X = F; R = 2 ' -deoxyr ibose-5'- FdUMP monophosphate Figure 4.1. A G e n e r a l i z e d s t r u c t u r e f o r U, dUrd, 5-FU, 5-FdUrd,- and 5-FdUMP. The most s t r i k i n g d i f f e r e n c e between these Raman s p e c t r a i s the presence of an intense peak at 1349 cm 1 f o r 5-FU, which i s absent i n the U spectrum. T h i s peak may be assigned to a C-F s t r e t c h i n g v i b r a t i o n on t h i s b a s i s , and because C-F s t r e t c h -ing f r e q u e n c i e s have p r e v i o u s l y been observed f o r model com-pounds i n t h i s region (11). F i n a l l y , a comparison of F i g u r e s 2.20b and 2.20c i n d i c a t e s a c l o s e resemblance between the s p e c t r a of p o l y c r y s t a l l i n e U and n e u t r a l U i n H nO. - 147 -I n f r a - r e d and u l t r a v i o l e t s p e c t r o s c o p i c measurements show t h a t the f i r s t i o n i z a t i o n f o r u r a c i l o c c u r s p r e f e r e n t i a l l y a t N-3 r a t h e r than a t N-1. 5 - F l u o r i n a t i o n l o w e r s t h i s f i r s t pK a. by 1.3 pH u n i t s (from 9.45 t o 8.15) and a l s o i n c r e a s e s the p r e -f e r e n c e -;f or N-3 i o n i z a t i o n ; 6 - f l u o r i n a t i o n i n c r e a s e s the p r o p o r -t i o n o f N-1 i o n i z a t i o n ( 1 0 ) . F i g u r e 2.21 shows Raman s p e c t r a o f 5-FU i n r ^ O a t s e v e r a l pH v a l u e s . As the pH i s i n c r e a s e d above n e u t r a l i t y , Raman s p e c t r a l changes c o i n c i d e w i t h the f i r s t and second d e p r o t o n a t i o n s t e p s ( p r< = 8.15, 13) ( 9 ) . Changes a t a pH >. 10 do not occur f o r the c o r r e s p o n d i n g n u c l e o s i d e s , because i n those c a s e s the N-1 p o s i t i o n i s s u b s t i t u t e d w i t h 2 - d e o x y r i -bose (see F i g u r e 4.1). 4.2.2. 2 ' - D e o x y u r i d i n e and 5 - F l u o r o - 2 ' - d e o x y u r i d i n e A comparison o f the Raman s p e c t r a f o r 5-FU i n F i g u r e 2.21 w i t h those f o r 5-FdUrd i n F i g u r e 2.22 i n d i c a t e s t h a t the i n t r o -d u c t i o n o f the sugar m o i e t y , which i t s e l f shows no o b s e r v a b l e Raman v i b r a t i o n s i n t h i s f r e q u e n c y range, a l t e r s s u b s t a n t i a l l y the v i b r a t i o n a l p r o p e r t i e s o f the 5-FU base. The e f f e c t s o f 5-f l u o r i n a t i o n on Raman p r o p e r t i e s o f the dUrd n u c l e o s i d e are e v i d e n t from a comparison o f the Raman s p e c t r a o f dUrd and 5-FdUrd i n f ^ O and D 20 shown i n F i g u r e s 2.22 and 2.23, r e s p e c t -i v e l y ; i n a l l c a s e s , the Raman v i b r a t i o n s from the base are con-f i n e d t o a s p e c t r a l range between 500 and 1750 cm Lor d and Thomas (12) have made r e l i a b l e assignments f o r s e v e r a l Raman - 148 -l i n e s i n aqueous (and D2O) s o l u t i o n s of u r i d i n e , p r o v i d i n g a . b a s i s f o r the present a n a l y s i s of 2 1 - d e o x y u r i d i n e and i t s 5-f l u o r i n a t e d d e r i v a t i v e . Double Bond S t r e t c h i n g Region: H^O In u r i d i n e , the s p e c t r a l r e g i o n between 1550 and 1750 cm c o n s i s t s p r i n c i p a l l y of v i b r a t i o n a l c o n t r i b u t i o n s from two c a r -bonyls and the C-5:C-6 double bond, with a s m a l l c o n t r i b u t i o n (in only) from a deformation mode of the a c i d i c N-3 proton (12). In H 20 i t i s not p o s s i b l e to r e s o l v e the i n d i v i d u a l c a r -bonyls nor the C-5:C-6 double bond s t r e t c h i n g v i b r a t i o n s . For dUrd i n H 20, the n e u t r a l form (Figure 2.22a) e x h i b i t s an intense peak at 1678 cm with a pronounced shoulder at 1626 cm Lord and Thomas (12) have e s t a b l i s h e d t h a t the 1676 cm peak i s predominantly due to c a r b o n y l s t r e t c h i n g , and the 1626 cm ^ shoulder to C-5:C-6 double bond s t r e t c h i n g v i b r a t i o n s . On d e p r o t o n a t i o n of the base a t N-3 (Figure 2.22c) , ,these two peaks are r e p l a c e d by l i n e s at approximately 1632 cm 1 and 1600 -1 cm For 5-FdUrd i n ^ 0 , the n e u t r a l form (Figure 2.22b) shows a strong broader peak centered at 1676 cm with a l e s s intense shoulder at approximately 1627 cm . In c o n t r a s t to dUrd, de-p r o t o n a t i o n (Figure 2.22d) of 5-FdUrd causes most of the i n t e n -s i t y to s h i f t to approximately 1604 cm l e a v i n g the remaining i n t e n s i t y a t approximately 1668 cm ^ (near the o r i g i n a l peak maximum before deprotonation) . • 149 -co in " " 1 1 " 1 1 " ' 1 1 1 1 1 " 1 " 1 1 1 1 " 1 1 < 1 1 1 ' " 1 1 1 1 1 " " 1 " 1 1 ' o o o o o o o o o o o o f > <0 *0 N (0 U) T - r - y r- y- r-_ C M " 1 . C M " 1 F i g u r e 4.2. Laser Raman c a r b o n y l s t r e t c h i n g region f o r 50 m i l l i m o l a r D2O s o l u t i o n s o f : (a) n e u t r a l dUrd (pH meter reading 5.8), (b) n e u t r a l 5FdUrd (pH meter reading 5.8), (c) a n i o n i c dUrd (pH meter 11.2), (d) a n i o n i c 5FdUrd (pH meter 11.1). These s p e c t r a were obtained as d e s c r i b e d i n s e c t i o n 2.8.2. - 150 -Double Bond S t r e t c h i n g Region: D 20 The much improved r e s o l u t i o n f o r the 1600-1700 cm ^ region i n D 20 permits a more d e f i n i t i v e a n a l y s i s i n terms of the p r i n -c i p l e resonance s t r u c t u r e s f o r n e u t r a l and a n i o n i c dUrd and 5-FdUrd. The Raman c a r b o n y l s t r e t c h i n g region of dUrd and 5-FdUrd (in D 20) are shown i n Figu r e 4.2. The p r i n c i p l e resonance s t r u c t u r e s f o r the n e u t r a l and a n i o n i c forms of these molecules are shown i n Fi g u r e 4.3. For n e u t r a l dUrd, the Raman spectrum of the ca r b o n y l region (Figure 4.2a) shows two v i b r a t i o n a l l y nonequivalent c a r b o n y l s , with C 2 = 0 at 1687 cm ^ and C 4 = 0 at 1653 cm ^, assigned by analogy to Urd (16,17). The lower frequency f o r the C-4 c a r -bonyl f a v o r s resonance c o n t r i b u t i o n s from l a and l b , c o n s i s t e n t with a lower i o n i z a t i o n p o t e n t i a l (by 1 e.v.) f o r the lone e l e c -tron p a i r of 0-4 compared to 0-2 (13). Furthermore, s t r u c t u r e l a i s favored over l b , because X-ray d i f f r a c t i o n - s h o w s that the C^-Cg bond i s sh o r t e r (1.34 angstroms) than the C^-C,- bond (1.44 angstroms) (14). For n e u t r a l 5-FdUrd, the Raman spectrum (Figure 4.2b) shows two v i b r a t i o n a l l y e q u i v a l e n t c a r b o n y l s at 1676 cm The C^=0 frequency has presumably i n c r e a s e d r e l a t i v e to the C 2 = 0 f r e -quency, compared to dUrd, because a - f l u o r o - s u b s t i t u t i o n i s known to s h i f t Raman c a r b o n y l s t r e t c h i n g to higher frequency i n model compounds (15). X-ray bond lengths (1.33 angstroms f o r C^-C^ and 1.44 angstroms f o r C^-Cj. i n 5-FdUrd) (6) d i s f a v o r - 151 -I la lb lc Id n e u t r a l l ib F i g u r e 4.3. The p r i n c i p l e resonance s t r u c t u r e s f o r n e u t r a l and a n i o n i c forms of dUrd (R = 2 ".-deoxyur i d i n e , X = 1E) and 5FdUrd (R = 2 1 - d e o x y u r i d i n e and X = 1 9 F ) . - 152 -s t r u c t u r e l b . Thus, the Raman equivalence of C2=0 and C^ = 0 i n F i g u r e 4.2b r e q u i r e s i n c r e a s e d c o n t r i b u t i o n s from s t r u c t u r e s l c and Id r e l a t i v e to Ia i n 5-FdUrd compared to dUrd. For dUrd, both Raman c a r b o n y l f r e q u e n c i e s s h i f t by the same amount (53 and 51 cm ^) on deprotonation (Figures 4.2a and 4.2c), suggesting comparable c o n t r i b u t i o n s from I l a and l i b i n the anion (perhaps b e t t e r represented as l i e ) . On the other hand, the (equivalent) Raman c a r b o n y l f r e q u e n c i e s f o r n e u t r a l 5-FdUrd become nonequivalent (1677 and 1608 cm "S on deprotona-t i o n (Figures 4.2b and 4.2d). T h i s nonequivalence almost c e r -t a i n l y f a v o r s s t r u c t u r e I l a over l i b or l i e i n the 5-FdUrd anion, because the 0^=0 i s c l o s e r to the s i t e of f l u o r i n a t i o n than i s C 2 = 0 ; thus, the C^ = 0 would be expected to experience the l a r -ger Raman s h i f t on d e p r o t o n a t i o n . In t h i s r e s p e c t , i t may be noted that the 1 3C-nmr chemical s h i f t of C-4 moves much f a r t h e r downfield on f l u o r i n a t i o n (by 147.5 Hz at 22.62 MHz) than does C-2 (43.0 Hz) (7). F i n a l l y , s i n c e f l u o r i n e s u b s t i t u t i o n i n c r e a s e s the frequency of a double bond s t r e t c h (11), the 1676 cm 1 envelope f o r n e u t r a l 5-FdUrd (Figure 4.2b) may i n c l u d e a c o n t r i b u t i o n from the C,--Cr double bond s t r e t c h . 6 V i b r a t i o n s below 1400 cm - 1: dUrd and 5-FdUrd i n H 2 0 and D 2 0 The most intense Raman l i n e f o r n e u t r a l dUrd i n H2O i s l o c a t e d at 1230 cm 1 (Figure 2.22a). This l i n e , which i s weak in i n f r a - r e d s p e c t r a , may be assigned, by analogy to u r i d i n e - 153 -( 1 2 ) , as o r i g i n a t i n g p r e d o m i n a n t l y from a c o n c e r t e d c a r b o n -n i t r o g e n s t r e t c h i n g o f the u r a c i l r i n g ( 1 6 ) , w i t h p r o b a b l e major c o n t r i b u t i o n from v i b r a t i o n s o f the two C-N bonds a s s o c i a t e d w i t h the p r o t o n a t e d N-3. T h i s i n t e r p r e t a t i o n i s c o n s i s t e n t w i t h the o b s e r v e d s e n s i t i v i t y of t h i s peak t o pH and d e u t e r a t i o n ( F i g u r e s 2.22a, 2.22c; 2.23a, 2.23c). For 5-FdUrd i n H 20, the same s p e c t r a l r e g i o n shows an i n t e n s e peak 1213 cm 1 and two s m a l l e r peaks a t 1236 cm 1 and 1268 cm 1 , and these peaks are a g a i n s e n s i t i v e t o pH and d e u t e r a t i o n ( F i g u r e s 2.22b, 2.22d; 2.23b, 2.23d). N e u t r a l dUrd g i v e s a m o d e r a t e l y i n t e n s e peak a t 1390 cm 1 i n H 20 (1398 cm""1 i n D 20) , which d i s a p p e a r s on N-3 d e p r o t o n a -t i o n . N e u t r a l 5-FdUrd does not show comparable Raman-active v i b r a t i o n s near t h i s f r e q u e n c y (the peak near 1360 cm 1 has a d i f f e r e n t o r i g i n — see b e l o w ) . The i n t e n s e l i n e l o c a t e d between 780 and 794 cm 1 , a s s i g n e d t o a r i n g - b r e a t h i n g type of m o t i o n , i s r e l a t i v e l y i n s e n s i t i v e t o d e p r o t o n a t i o n , d e u t e r a t i o n , or f l u o r i n a t i o n o f dUrd ( F i g u r e s 2.22 and 2.23). S i m i l a r l y , the weak l i n e a t 626-630 c m - 1 a l s o appears i n s e n s i t i v e t o d e p r o t o n a t i o n , d e u t e r a t i o n , or f l u o r i n a -t i o n o f dUrd. There i s a l i n e a t 560 cm 1 i n H 20 f o r dUrd (and a t 690 cm 1 f o r 5-FdUrd) which s h i f t s t o h i g h e r f r e q u e n c y (by 27 cm 1 f o r dUrd and by 13 cm 1 f o r 5-FdUrd) on d e p r o t o n a t i o n , and a s i m i l a r e f f e c t i s seen i n D~0 ( F i g u r e s 2.23). - 154 -F i n a l l y there i s one prominent peak f o r 5-FdUrd (1360 cm at n e u t r a l pH i n ^ O ; 1348 cm ^ on depr o t o n a t i o n i n E^O) i n Fi g u r e s 2.22b and 2.22d, which i s r e l a t i v e l y u n a f f e c t e d by d e u t e r a t i o n (Figure 2.23b; 2.23d). We t e n t a t i v e l y a s s i g n t h i s peak to a C-F s t r e t c h , s i n c e there i s no corresponding peak f o r the u n f l u o r i n a t e d dUrd, and because C-F s t r e t c h i n g f r e q u e n c i e s have been observed f o r other compounds i n t h i s r e gion (11). 4.2.3 5-Fluoro-2'-deoxyuridine (5-FdUrd) and 5-Fluoro- 2'-deoxyuridine-5'-monophosphate (5-FdUMP) The l a s e r Raman s p e c t r a of 5-FdUrd and 5-FdUMP, shown i n Fig u r e 2.24, e x h i b i t no major changes i n the Raman v i b r a t i o n s due to the 5'-phosphate group. The 976 cm 1 l i n e , at pH > 7, i n d i c a t e d i n F i g u r e 2.24c i s due to a symmetric s t r e t c h i n g v i b r a -_2 t i o n of the PO^ group (17). The o n l y other d e t e c t a b l e a l t e r a -t i o n which may be due to the phosphate group i s a broadening of the s p e c t r a l envelope f o r the concerted r:ing v i b r a t i o n a t 786 cm , shown i n Fi g u r e 2.24a. 4.2.4 Concluding Remarks To summarize t h i s s e c t i o n , the new Raman data i n d i c a t e that one important e f f e c t of 5 - f l u o r i n a t i o n on the n e u t r a l form of 2'-deoxyuridine i s to make the two ca r b o n y l s more e q u i v a l e n t by i n c r e a s i n g the C^=0 s t r e t c h i n g frequency r e l a t i v e to the C2=0 s t r e t c h , and the e f f e c t of 5-f l u o r i n a t i o n on the a n i o n i c form i s to favor s t r u c t u r e s with more charge d e n s i t y at C^=0 r e l a t i v e to C 9 = 0 than i n unf l u o r i n a t e d 2 '-deoxyur i d i n e . A l l - 155 -peaks ^observed i n s o l u t i o n s of n e u t r a l 5-FU, 5-FUrd, and 5-FUMP are a l s o present f o r the p o l y c r y s t a l l i n e forms of each s p e c i e s . A new prominent p H - s e n s i t i v e peak at 1360 cm 1 (5-FUrd, H^O, n e u t r a l pH), i n s e n s i t i v e to d e u t e r a t i o n at N-3, i s t e n t a t i v e l y assigned to a C-F s t r e t c h . Other major peaks have been assigned by analogy to p r e v i o u s l y analyzed s p e c t r a of u r i d i n e (12). A major c o n s i d e r a t i o n of t h i s study was to evaluate the e f f e c t s o f 5-f l u o r o - -.substitution on the peak i n t e n s i t i e s o f the u r a c i l base r i n g v i b r a t i o n s . T h i s was a p p l i e d to the i n -t e r p r e t a t i o n of the FU-5SrRNA Raman spectrum. The 782 cm 1 and 1230 cm 1 l i n e s of dUrd i n H 20 are reduced by at l e a s t 40% and 87% r e s p e c t i v e l y due to 5 - f l u o r o - s u b s t i t u t i o n (Figure 2.22a and 2.22b). These two Raman l i n e s w i l l be con s i d e r e d i n the i n t e r p r e t a t i o n of the FU-5SrRNA Raman spectrum shown i n s e c t i o n 4.3. - 156 -4.3 Las e r Raman S p e c t r o s c o p y o f N-5SrRNA and FU-5SrRNA As p r e v i o u s l y mentioned changes i n the i n t e n s i t i e s o f v a r i o u s Raman l i n e s o f an RNA spectrum are an i n d i c a t i o n o f c o n f o r m a t i o n a l change. Most o f the Raman l i n e s due t o the p u r i n e and p y r i m i d i n e bases are hypochromic ( 3 ) . That i s , they d e c r e a s e i n i n t e n s i t y w i t h an i n c r e a s e i n the s t a c k i n g e f f i c i e n c y o f the bases. T h i s o b s e r v e d h y p o c h r o m i c i t y i s o f t e n d i f f i c u l t t o i n t e r p r e t because o f e x t e n s i v e o v e r l a p from the v i b r a t i o n a l c o n t r i b u t i o n s o f d i f f e r e n t bases. T h i s i s e s -p e c i a l l y t r u e o f the s p e c t r a l r e g i o n between 1200-1600 cm ^ (3) . A l i n e a t 725 cm i s due e x c l u s i v e l y t o adenine r e s i d u e s . T h i s l i n e i s hypochromic s i n c e a decrease i n i t s i n t e n s i t y c o r -responds t o more e f f i c i e n t s t a c k i n g o f these r e s i d u e s (18-21). C o n v e r s e l y , the 670 cm l i n e which i s due e x c l u s i v e l y t o guanine i s r e v e r s e hypochromic (22,23) s i n c e i n c r e a s e d s t a c k -i n g i n t e r a c t i o n s cause an i n c r e a s e i n i t s i n t e n s i t y . Another l i n e o f d i a g n o s t i c u t i l i t y i s the 814 cm l i n e which has been a s s i g n e d t o the symmetric s t r e t c h i n g o f o r d e r e d p h o s p h o d i e s t e r bonds of the r i b o p h o s p h a t e backbone (18-21). An i n c r e a s e i n the i n t e n s i t y o f t h i s l i n e i s i n d i c a t i v e o f i n c r e a s e d r e g u l a r -i t y of the phosphate backbone. Together the above t h r e e Raman l i n e s are p a r t i c u l a r l y u s e f u l because they are each due t o a s i n g l e v i b r a t i o n a l component. S i n c e they a r e not c o m p l i c a t e d from o v e r l a p p i n g c o n t r i b u -t i o n s o f d i f f e r e n t o r i g i n s the 670 cm ^, 725 cm , and 814 cm ^ - 157 -Raman l i n e s are the most u s e f u l i n d i c a t o r s of RNA conformation. Phe -1 In yeast tRNA the 670 cm l i n e undergoes almost a 3 f o l d decrease when the guanine bases go from a completely stacked c o n f i g u r a t i o n to an unstacked form (22,23). Conversely, the 725 cm 1 l i n e i n c r e a s e s about 12% when the p a r t i a l l y stacked adenine r e s i d u e s become unstacked (3). The 814 cm 1 l i n e i s a measure of the h e l i c a l order of the ribophosphate backbone (18-21). I t s i n t e n s i t y i s i n f l u e n c e d by the geometry of the C-O-P-O-C l i n k a g e s and an i n c r e a s e i n i n t e n s i t y i m p l i e s g r e a t e r h e l i c a l o r d e r . A comparison of the Raman s p e c t r a of N-5SrRNA before and a f t e r d i a l y s i s are shown in F i g u r e 2.25. T h i s shows that con-f o r m a t i o n a l changes have taken p l a c e . In Table 2.3 the peak i n t e n s i t i e s , normalized to the i n v a r i a n t 1100 cm 1 l i n e , are g i v e n . The observed i n c r e a s e i n the i n t e n s i t y of the 670 cm 1 l i n e by about 12% i s an i n d i c a t i o n of more e f f i c i e n t s t a c k i n g of the G bases due to d i a l y s i s a g a i n s t magnesium. S i m i l a r l y , A s t a c k i n g i s improved s i n c e there i s a decrease of about 19% a f t e r d i a l y s i s . Both samples e x h i b i t comparable r e g u l a r i t y i n t h e i r ribophosphate backbone as e v i d e n t from i d e n t i c a l i n t e n -s i t y values f o r the 814 cm 1 l i n e . The 785 cm 1 l i n e decreases by almost 25%. T h i s l i n e i s due to the combined e f f e c t s of c y t o s i n e and u r i d i n e r e s i d u e s and reduces in i n t e n s i t y with improved s t a c k i n g i n t e r a c t i o n s of these r e s i d u e s . Consequent-l y , the s u b s t a n t i a l r e d u c t i o n i s an i n d i c a t i o n of more e f f i c i e n t - 158 -s t a c k i n g of c y t o s i n e and/or u r i d i n e bases. The Raman l i n e s above 1200 cm ^ are a l s o s e n s i t i v e to s t a c k i n g i n t e r a c t i o n s and decrease with improved s t a c k i n g (3). However, the o v e r l a p p i n g of the v i b r a t i o n a l c o n t r i b u t i o n s from d i f f e r e n t bases hinder i n t e r p r e t a t i o n . C l e a r l y , a l t e r a t i o n s i n s t a c k i n g i n t e r a c t i o n s due to d i a l y s i s a g a i n s t magnesium are apparent i n the s p e c t r a l region between 1200 cm 1 and 1600 cm ^ (see Table 12 . 5 ) • The Raman s p e c t r a of the d i a l y s e d sample of N-5SrRNA and that obtained by Chen e t . a l . are comparable. D i f f e r e n c e s i n ta b u l a t e d i n t e n s i t y values are a t t r i b u t a b l e to t h e i r s e l e c t i o n of d i f f e r e n t b a s e l i n e s . Measurement of the r e l a t i v e i n t e n s i -t i e s of the 814 cm , 785 cm , 725 cm , and 670 cm ^ l i n e s using b a s e l i n e s c o n s i s t e n t with t h i s work g i v e s values of 1.60, 2.32, 0.68 and 0.72 r e s p e c t i v e l y ; these agree f a v o r a b l y with those val u e s shown i n Table 2.5!. The Raman s p e c t r a of the d i a l y s e d sample of N-5SrRNA and FU-5SrRNA are s i m i l a r . These s p e c t r a are shown i n Fig u r e 2.26 and i n t e n s i t i e s of Raman l i n e s , r e l a t i v e to the 1100 cm l i n e , are given i n Table '2.5. The i n t e n s i t i e s of the 670 cm ^ l i n e s , due to guanine r e s i d u e s , are almost i d e n t i c a l ; an i n -d i c a t i o n of comparable s t a c k i n g of t h i s r e s i d u e . Some destack-ing of adenine r e s i d u e s i s apparent s i n c e the 725 cm l i n e i n c r e a s e s f o r the 5-FU c o n t a i n i n g sample. The i n t e n s i t y of the 814 cm ^ l i n e i s s l i g h t l y g r e a t e r f o r FU-5SrRNA which may i n d i c a t e a s l i g h t l y more ordered ribophosphate backbone. - 159 -The Raman s p e c t r a of dUrd and 5-FdUrd shown i n Fig u r e 2.22 i n d i c a t e r e d u c t i o n i n the i n t e n s i t i e s of both the 1234 cm 1 and 787 cm 1 l i n e s of u r i d i n e due to 5 - f l u o r o - s u b s t i t u -t i o n . In the case of the 1234 cm 1 l i n e the r e d u c t i o n i s ap-proximately 87%. The 787 cm 1 l i n e i s reduced by about 40%. These r e d u c t i o n s are expected to a f f e c t the 785 cm 1 and 1242 cm 1 l i n e s i n p r o p o r t i o n to the r e l a t i v e number of 5-FU r e s i -dues i n FU-5SrRNA. 5SrRNA has 20 u r a c i l r e s i d u e s and 36 c y t o -sine r e s i d u e s . The u r i d i n e r e s i d u e s c o n t r i b u t e about 25% of the 785 cm 1 l i n e i n t e n s i t y (see Fig u r e 2.22a) while the c y t i d i n e r e s i d u e s c o n t r i b u t e approximately 75% (24). Assuming that there i s 100% replacement of u r i d i n e by 5 - f l u o r o u r i d i n e and that c y t i d i n e , u r i d i n e , and 5 - f l u o r o u r i d i n e maximum i n t e n s i t i e s f a l l at the same Raman frequency (785 cm then a r e d u c t i o n i n the u r i d i n e c o n t r i b u t i o n by about 60% would be expected (see F i g -ures 2.22a and 2.22b). In N-5SrRNA the i n t e n s i t y of the 785 cm 1 l i n e i s 2.21. T h i s means that for N-5SrRNA 0.55 i s due to u r i d i n e (25%) and 1.66 i s from c y t i d i n e (75%). In an FU-5SrRNA sample, where a l l the u r i d i n e s are re p l a c e d by 5 - f l u o r o u r i d i n e , the 5 - f l u o r o u r i d i n e c o n t r i b u t i o n to the 785 cm 1 l i n e w i l l be 60% l e s s than f o r normal u r i d i n e or equal to 0.22. This g i v e s a t o t a l i n t e n s i t y f o r the FU-5SrRNA sample of approximately 0.22 + 1.66 = 1.88; a r e d u c t i o n o f about 15%. A r e d u c t i o n o f 15% r e p r e s e n t s the absolute maximum decrease i n i n t e n s i t y f o r the 785 cm 1 l i n e s i n c e i t assumes 100% replacement of u r i d i n e - 1 6 0 -by 5 - f l u o r o u r i d i n e . The e x p e c t e d 5 - f l u o r o u r i d i n e i n c o r p o r a -t i o n was a b o u t 85% ( 2 5 ) . Assum i n g 85% r e p l a c e m e n t by 5 - f l u o r o -u r i d i n e , a r e d u c t i o n i n i n t e n s i t y o f t h e 785 cm l i n e o f a b o u t 12% would be e x p e c t e d . In T a b l e 2.3 the r e l a t i v e i n t e n s i t i e s f o r t h e 785 c m - 1 l i n e s o f N-5SrRNA and FU-5SrRNA a r e 2.21 and 2.10 r e s p e c t i v e l y . T h i s c o r r e s p o n d s t o a 5% r e d u c t i o n due t o 5 - f l u o r o u r i d i n e s u b s t i t u t i o n . D e v i a t i o n f r o m t h e e x p e c t e d v a l u e o f a b o u t 12% r e d u c t i o n c o u l d be due t o l e s s e x t e n s i v e i n -c o r p o r a t i o n t h a n e x p e c t e d o r h y p o c h r o m i c e f f e c t s due t o s t a c k -i n g i n t e r a c t i o n s between 5-FU and n e i g h b o r i n g b a s e s . F o r t h e 1 2 4 2 cm ^ l i n e u r i d i n e r e s i d u e s c o n t r i b u t e a b o u t 26% ( F i g u r e 2 . 2 2 a ) , c y t i d i n e r e s i d u e s a b o u t 69% ( 2 4 ) , and ade-n i n e r e s i d u e s a b o u t 5% ( 2 6 ) . In N-5SrRNA t h e peak i n t e n s i t y o f the 1242 cm ^ l i n e i s 1.23 o f w h i c h 26% (0.32) i s due t o u r i -d i n e . A r e d u c t i o n o f 87% r e d u c e s the u r i d i n e " c o n t r i b u t i o n t o 0.04. T h i s g i v e s an e x p e c t e d l i n e i n t e n s i t y f o r the FU-5SrRNA sample o f 0.95 o r a r e d u c t i o n when compared t o N-5SrRNA o f a b o u t 23%. The 1242 c m - 1 l i n e s o f N-5SrRNA and FU-5SrRNA have the same i n t e n s i t i e s . In t h e above argument i t was assumed t h a t t h e maximum peak i n t e n s i t i e s f o r u r i d i n e , a d e n i n e , and 5 - f l u o r o -a d i n i n e o c c u r e d a t t h e same f r e q u e n c y (1242 cm "*") . A c t u a l l y the 1242 cm ^ l i n e i s r e a l l y a s u p e r p o s i t i o n o f the 1234 cm u r i d i n e l i n e and a 1251 cm l i n e due t o a d e n i n e and c y t i d i n e ( 3 ) . C o m p a r i s o n o f l i n e w i d t h s a t h a l f , t h e i n t e n s i t y o f the 1242 cm ^ l i n e i n d i c a t e t h a t f o r N-5SrRNA i t i s much l a r g e r ; - 161 -40 cm as compared to 24 cm for FU-5SrRNA. This i s a t t r i -buted to replacement of u r i d i n e by 5 - f l u o r o u r i d i n e . Other sma l l changes i n the s p e c t r a l region between 1200-1600 cm 1 are e v i d e n t . These d i f f e r e n c e s must be a t t r i b u t e d to a l t e r a t i o n s i n conformation r e s u l t i n g from 5 - f l u o r o u r i d i n e i n c o r p o r a t i o n but i n t e r p r e t a t i o n i s hindered by e x t e n s i v e over-l a p p i n g of v i b r a t i o n a l c o n t r i b u t i o n s from v a r i o u s bases (3). Three major c o n c l u s i o n s can be obtained from the compari-son of the Raman s p e c t r a of N-5SrRNA and FU-5SrRNA. F i r s t , the t o t a l base s t a c k i n g i s l a r g e l y u n a f f e c t e d by 5-FU s u b s t i t u t i o n and t h e r e f o r e the conformation of the two molecules must be f a i r l y s i m i l a r . Second, the amount of G-stacking i s u n a f f e c t e d by s u b s t i t u t i o n c o n f i r m i n g a s i m i l a r conformation. L a s t l y , minor d i f f e r e n c e s i n the s p e c t r a of FU-5SrRNA and N-5SrRNA are that the backbone order f o r the FU s p e c i e s i s s l i g h t l y g r e a t e r and the adenine and u r i d i n e s t a c k i n g are s l i g h t l y l e s s i n FU-5SrRNA. The in c r e a s e i n backbone order may be a r e s u l t of i n -creased s t r e n g t h of bonding of a d e n i n e - u r i d i n e p a i r s due to the presence of the e l e c t r o n e g a t i v e f l u o r i n e group (27). The de-crease i n adenine and u r i d i n e s t a c k i n g i s l i k e l y a r e s u l t of decreased s t a c k i n g e f f i c i e n c y f o r a d e n i n e : 5 - f l u o r o u r i d i n e p a i r s . The presence of the f l u o r i n e may prevent e f f i c i e n t o v e r l a p of the a d e n i n e : 5 - f l u o r o u r i d i n e base p a i r e l e c t r o n clouds with neighboring base p a i r s . - 162 -The l a s e r Raman spectrum of FU-5SrRNA was obtained with-out d i a l y s i s of the sample a g a i n s t magnesium. The spectrum's high degree of order, comparable t o N-5SrRNA a f t e r d i a l y s i s , i s a t t r i b u t e d to the 5 - f l u o r o u r a c i l ' r e s i d u e s . The d i a l y s i s a g a i n s t magnesium i s b e l i e v e d to renature N-5SrRNA (28). The d i a l y s i s of the FU-5SrRNA sample r e s u l t e d i n unacceptably high f l u o r e s c e n c e background which prevented the obtainment of a spectrum. The above r e s u l t s suggest t h a t 5-FU s u b s t i t u t i o n i n 5SrRNA i s expected to cause minimal p e r t u r b a t i o n o f s t r u c t u r e and c o n c l u s i o n s r e s u l t i n g from a study of FU-5SrRNA s t r u c t u r e should be a p p l i c a b l e to N-5SrRNA. - 163 -REFERENCES: CHAPTER 4 1. G.J. Thomas and K.A. Hartman, B i o c h i m . B i o p h y s . A c t a . 312, (1973) . 2. G.J. Thomas, M.C. Chen, and K.A. Hartman, B i o c h i m . B i o p h y s .  A c t a . 324, 37. 3. M.C. Chen, R. Giege', R.C. L o r d , and A. R i c h , B i o c h e m i s t r y 14 , 4385 (1975) . 4. K.A. Hartman, R.C. L o r d , and G.J. Thomas i n P h y s i c o - c h e m - i c a l P r o p e r t i e s o f N u c l e i c A c i d s , J . Duchesne, E d . , A c a d e m i c P r e s s , London, V o l . 1 (1973); page 1. 5. L. F a l l o n , A c t a . C r y s t . B29, 2549 ( 1 9 7 3 ) . 6. D.R. H a r r i s and W.M. M a c i n t y r e , B i o p h y s . J . 4, 203 ( 1 9 6 4 ) . 7. A.R. T a r p l e y and J.H. G o l d s t e i n , J . Am. Chem. Soc. 93, 3573 ( 1 9 7 1 ) . 8. R . J . C u s h l e y , I . Wempen, and J . J . Fox, J . Am. Chem. S o c . 90, 709 (1968) . 9. I . Wempen and J . J . Fox, J . Am. Chem. S o c . 86, 2474 (1964). 10. K.L. W i e r z c h o w s k i , E . L i t o n s k a , and D. Shogan, J . Am.  Chem. Soc. 8J7 , 4621 (1965) . 11. F.U. D o l l i s h , W.A. F a t e l e y , and F . F . B e n t l e y , i n C h a r a c - t e r i s t i c Raman F r e q u e n c i e s o f O r g a n i c Compounds, John W i l e y & Sons, I n c . , New York (1974); pages 67-68. 12. R.C. L o r d and G.J. Thomas, S p e c t r a c h i m . A c t a . 231, 2551 (1967) . 13. A. Padua, P.R. L e B r e t o n , R . J . D i n e r s t e i n , and J.N.A. R i d -y a r d , B i o c h e m . B i o p h y s . Res. Comm. 60 , 1262 ( 1 9 7 4 ) . 14. A. Rahman and H.R. W i l s o n , A c t a . C r y s t . B28, 2260 (1972). 15. L . J . B e l l a m y , i n The I n f r a - r e d S p e c t r a o f Complex M o l e - c u l e s , Chapman and H a l l , London; pages 1570159. 16. Y. N i s h i m u r a , H. Haruyama, K. Nomura, A.Y. H i r a k a w a , and M. T s u b o i , B u l l . Chem. S o c . J a p a n 52, 1340 ( 1 9 7 9 ) . - 164 -17. Reference 4, V o l . 2; pages 111-124. 18. W.L. P e t i c o l a s , i n Advances in Raman Spectroscopy, J.P. Mathieu, ed., Heyden & Son, New York (1972); pages 285-295. 19. G.J. Thomas, i n V i b r a t i o n a l S pectra and S t r u c t u r e , J.R. Durig, ed. , E l e s e v i e r , New York (1975); pages 239-315. 20. G.J. Thomas and K.A. Hartman, Biochim. Biophys. A c t a . 312, 311 (1973). 21. M.C. Chen and G.J. Thomas, Biopolymers 13, 615 (1974). 22. J.E. Ladner, A. Jack, J.D. Robertus, R.S. Borwn, D. Rhodes, B.F.C. C l a r k , and A. Klug, Proc. Nat. Acad. S c i . USA 72, 4414 (1975). 23. S.H. Kim, F.L. Suddath, G.J. Q u i g l e y , A. McPherson, J.L. Sussman, A.H.J. Wang, N.C. Seeman, and A. R i c h , Science  185, 435 (1974). 24. T. O'Connor, C. Johnson, and W.M. S c o v e l l , Biochim. B i o - phys. A c t a . 447, 484 (1976). 25. I. K a i s e r , B i o c h e m i s t r y 9, 569 (1970). 26. T. O'Connor, C. Johnson, and W.M. S c o v e l l , Biochim. B i o - phys. A c t a . 447, 495 (1976). 27. L. P a u l i n g , i n The Nature of the Hydrogen Bond, C o r n e l l U n i v e r s i t y Press, New York (1972); page 452. 28. M. Aubert, J.F. S c o t t , M. Reynier, and R. Monier, Proc. Nat. Acad. S c i . USA 61, 292 (1968). - 165 -CHAPTER 5 CONCLUDING REMARKS From t h i s study i t can be concluded that 1 9F-nmr s p e c t r o -scopy and l a s e r Raman spectroscopy can be used to i n t e r p r e t c o n f o r m a t i o n a l p r o p e r t i e s of 5SrRNA (or FU-5SrRNA). The major advantage of both these p h y s i c a l techniques i s that they pro-vide i n f o r m a t i o n about molecular conformation i n an aqueous environment. T h i s i s apparent from s p e c t r a l changes which r e -s u l t when c o n d i t i o n s of the molecular environment are a l t e r e d . The 1 9F-nmr s p e c t r a of FU-5SrRNA presented in t h i s work i n d i c a t e a number of important f e a t u r e s . The spectrum at 254 MHz c o n s i s t s of 8 d i s t i n c t peaks and 2 shoulders with a chemical s h i f t range of approximately 8 p.p.m.. Together they represent f l u o r i n e resonances from about 20 5 - f l u o r o u r a c i l r e s i d u e s . The most exposed r e s i d u e s are assigned on the b a s i s of the resonant frequency of the 5 - f l u o r o - 2 ' - d e o x y u r i d i n e monophosphate monomer and the heat d e n a t u r e a t i o n of FU-5SrRNA. The remaining f l u o r i n e peaks (about 70% of the t o t a l ) are b e l i e v e d to be due to b u r i e d 5 - f l u o r o u r a c i l r e s i d u e s . The T^ values for the i n d i v i d u a l peaks are a l s o determined; a l l are s h o r t (between 0.3 - 0.4 seconds), an i n d i c a t i o n of a r i g i d molecular s t r u c t u r e . T h i s i s i n con-t r a s t to the T^ value of the 5 - f l u o r o - 2 1 - d e o x y u r i d i n e monomer which i s approximately 5 seconds. Comparison of t h e o r e t i c a l and experimental ^ F ^ H n u c l e a r Overhauser enhancements - 166 -demonstrates d e f i n i t i v e l y that v i r t u a l l y a l l the l a b e l e d u r a -c i l s are bound r i g i d l y to the macromolecular frame with a r o -t a t i o n a l c o r r e l a t i o n time of about 10 nsec or l o n g e r . Since these u r a c i l r e s i d u e s are widely d i s t r i b u t e d throughout the n u c l e o t i d e sequence, i t may be concluded that the e n t i r e FU-5SrRNA s o l u t i o n s t r u c t u r e i s h i g h l y r i g i d . T h i s l a t t e r e x p e r i -ment a l s o confirms t h a t r e l a x a t i o n i s p u r e l y d i p o l a r s i n c e almost f u l l Overhauser n u l l i n g i s observed. From the l a s e r Raman spectroscopy data two important con-c l u s i o n s can be a s c e r t a i n e d . F i r s t , 5 - f l u o r o - s u b s t i t u t i o n a l t e r s c e r t a i n v i b r a t i o n a l p r o p e r t i e s of the u r a c i l base. The i n t e n s i t i e s of the 782 cm 1 and 1230 cm 1 l i n e s of 2'-deoxyuri-dine i n H^O ( n e u t r a l form) are reduced by at l e a s t 40% and 87%, r e s p e c t i v e l y , due to 5 - f l u o r o - s u b s t i t u t i o n . A new prominent pH- s e n s i t i v e peak at 1360 cm 1 ( 5 - f l u o r o - 2 1 - d e o x y u r i d i n e , f^O, n e u t r a l pH), i n s e n s i t i v e to d e u t e r a t i o n at N-3, i s t e n t a t i v e l y assigned to a C-F s t r e t c h . An a d d i t i o n a l e f f e c t of 5 - f l u o r i n a -t i o n on the n e u t r a l form of 2 1 - d e o x y u r i d i n e i s to cause an i n -crease i n the C^=0 s t r e t c h i n g frequency r e l a t i v e to the C2=0 s t r e t c h . The e f f e c t of 5 - f l u o r i n a t i o n on the a n i o n i c form i s to favor more charge d e n s i t y at C^=0 r e l a t i v e to C2=0. Sec-ond, from comparison of N-5SrRNA s p e c t r a with those of FU-5SrRNA i t seems e v i d e n t that 5 - f l u o r o u r a c i l s u b s t i t u t i o n i n 5SrRNA causes o n l y minimal p e r t u r b a t i o n of s t r u c t u r e and, thus, con-c l u s i o n s r e s u l t i n g from a study of FU-5SrRNA s t r u c t u r e should be a p p l i c a b l e to N-5SrRNA. - 167 -Recent i n f r a r e d spectroscopy of n a t i v e E. c o l i 5SrRNA i n d i c a t e s t h a t a l l of i t s 20 u r a c i l s are base p a i r e d (6). Four of the u r a c i l s were shown to be t e r t i a r y and the remaining 16 secondary. In the numerous proposed s t r u c t u r e s of E. c o l i 5SrRNA the number of base p a i r s i n v o l v i n g u r a c i l range from 4 to 14. The comparison of the i n f r a r e d spectrum of E. c o l i 5SrRNA at 52 °C, where t e r t i a r y base p a i r s are presumed d i s -r upted, with computer simulated s p e c t r a of v a r i o u s proposed secondary s t r u c t u r e s of 5SrRNA are shown in F i g u r e s 5.1 - 5.4. These r e s u l t s i n d i c a t e that only models in F i g u r e s 5. l e , 5.2b, 5.3c, 5.3f, and 5.4b have simulated s p e c t r a s i m i l a r to the e x p e r i m e n t a l l y obtained spectrum at 52 °C. In Figure 5.5 simulated s p e c t r a f o r models where t e r t i a r y i n t e r a c t i o n s are proposed were compared with the IR spectrum of E. c o l i 5SrRNA at 20 °C. Only the simulated spectrum in F i g u r e 5.5a f i t s the experimental spectrum. In a l l cases f o r both the 52 °C and 20 °C s p e c t r a the proposed models which best f i t the e x p e r i -mental spectrum have the most ext e n s i v e A-U base p a i r s . The 1 9F-nmr study tends to c o r r o b o r a t e these r e s u l t s . At l e a s t 70% of the 5 - f l u o r o u r a c i l r e s i d u e s were shown to experience secon-dary or t e r t i a r y e f f e c t s . T h i s f i t s w e l l with models proposed by Cantor (7) and Luoma and M a r s h a l l (10 A-U and 6 G-U p a i r s ) (4) . In c o n c l u s i o n , v i r t u a l l y a l l p r e v i o u s l y proposed secondary s t r u c t u r a l models f o r p r o k a r y o t i c 5SrRNA (1-2) have a la r g e - 168 -F i g u r e 5.1. Simulated i n f r a r e d s p e c t r a f o r E. c o l i 5SrRNA s t r u c t u r a l models (—) i n comparison to the experimental spec-trum recorded at 52 C (...) (6) . - 169 -Figure 5.2. Simulated i n f r a r e d s p e c t r a f o r E. c o l i 5SrRNA s t r u c t u r a l models (—) i n comparison to the experimental spectrum recorded at 52°C (...)(6). - 170 -Figure 5.3. Simulated i n f r a r e d s p e c t r a f o r E. c o l i 5SrRNA s t r u c t u r a l models (—) i n comparison to the experimental spectrum recorded at 52°C (...)(6). F i g u r e 5.4. Simulated i n f r a r e d s p e c t r a f o r E. c o l i 5SrRNA s t r u c t u r a l models (—) i n comparison to the experimental spectrum recorded at 52°C (...)(6). - 172 -F i g u r e 5.5. Simulated i n f r a r e d s p e c t r a f o r those E. c o l i 5SrRNA s t r u c t u r a l models in which t e r t i a r y i n t e r a c t i o n s have been pro-posed (—) in comparison to the experimental spectrum recorded at 20°C ( ) (6) . - 173 -f r a c t i o n ( u s u a l l y more than h a l f ) of the u r a c i l r e s i d u e s l e f t u npaired. In c o n t r a s t , the present t h e o r e t i c a l and experimen-t a l 1 9F-nmr and 1 9 F - 1 H nuclear Overhauser enhancement r e s u l t s c o n v i n c i n g l y demonstrate t h a t e s s e n t i a l l y a l l f l u o r o u r a c i l r e s i d u e s i n E. c o l i 5SrRNA are bound f i r m l y to a r i g i d macro-molecular frame i n s o l u t i o n . These r e s u l t s are c o n s i s t e n t with Raman data (3) showing a high degree of o v e r a l l b a s e - p a i r i n g and RNA A - h e l i x content i n n a t i v e E. c o l i 5SrRNA. Both the Raman and 1 9F-nmr data support the most r e c e n t l y proposed " c l o -v e r l e a f " secondary s t r u c t u r e f o r p r o k a r y o t i c 5SrRNA (4-5). - 17 "4 -REFERENCES: CHAPTER 5 V.A. Erdman, Prog. N u c l e i c A c i d Res. 18 , 45 (1976) . V.A. Erdman, B. Appel, M. Digweed, D. Kluwe, S. Lorenz, A. Luck, A. S c h r e i b e r , and L. Schuster, i n The Genetic and  E v o l u t i o n a r y Aspects of T r a n s c r i p t i o n a l and T r a n s l a t i o n a l  Apparatus, Kondansha S c i e n t i f i c , Tokyo, 1979. M.C. Chen, R. Giege, R.C. Lord, and A. Ri c h , B i o c h e m i s t r y 17 , 3134 (1978 ) . G.A. Luoma and A.G. M a r s h a l l , Proc. N a t l . Acad. S c i . U.S.A. 75, 4901 (1978). G.A. Luoma and A.G. M a r s h a l l , J . Moi. B i o l . 125, 95 (1978). B. Appel, V.A. Erdman, J . S t u l z , and Th. Ackermann, Nuc. A c i d . Res. 7, 1043 (1979). C. R. Cantor, Nature 216, 513 (1967). 

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