Evidence for hydrogen bonds in a ribonucleoprotein

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Evidence for hydrogen bonds in a ribonucleoprotein I n view of evidence t h a t the ribonucleoproteins represent a stage in the process of protein biosynthesis 1, the n a t u r e of the protein-ribonucleic acid (RNA) linkage is of considerable biological interest. While the b o n d i n g between nucleic acid and p r o t e i n in the deoxyribonucleoproteins is considered to be electrostatic 2, it has been noted t h a t certain properties of the ribonucleoproteins are n o t those of pure electrostatic complexes, and it has been suggested t h a t h y d r o g e n bonds m a y be involved 3,4,5. I n a preliminary investigation of this problem, ribonucleoproteins, before and after t r e a t m e n t w i t h urea, h a v e been subjected to zone electrophoresis in starch over the p H range 3.5-1o.6 at ionic s t r e n g t h o.o5-o.I. The initial findings indicate t h a t h y d r o g e n b o n d s play an i m p o r t a n t role in linking the R N A and protein. The nucleoprotein was p r e p a r e d from E s c h e r i c h i a coli at 0-3 ° in o . o o 5 M n e u t r a l p h o s p h a t e buffer containing o.00075 M CaC12S. Logarithmic phase cells were g r o u n d w i t h glass p o w d e r a n d centrifuged for i h at 20,000 × g. The s u p e r n a t a n t was treated w i t h 1.25 % sodium desoxycholate, and one volume of ethanol was added. The material was t h e n precipitated twice with b a r i u m acetate (being redissolved with buffer containing o.oi M Na=SO4) and 3 or 4 times with (NH4)2SO4, ending with the fraction sedimenting b e t w e e n o. 3 and o.5 saturation. The final p r e p a r a t i o n s were dialyzed against either buffer or water. T h e y were free of deoxyribonucleic acid, were stable in the cold for at least a week, and were attacked b y crystalline pancreatic ribonuclease at 29 ° and at 0-3 °. T h e y contained 60-70 % R N A b y weight (RNA as per cent of R N A plus protein), corresponding to a molar ratio of ribonucleotide to a m i n o acid residues of a b o u t 0.5-0. 7. Thus, the R N A c o n t e n t was equivalent to the highest values previously reported for ribonucleoproteins from o t h e r sources 4,L8. There was present, however, a minor protein c o m p o n e n t which was electrophoretically distinct from the nucleoprotein (Figs. IA and 2A).

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Fig. i. Zone electrophoresis in s t a r c h at o-3 °. Sodium tu p h o s p h a t e , p H 9.o, ionic s t r e n g t h o.I ; 2.75 h a t 435-395 "~ 0.5volts followed b y 3 h a t 35o-34 o volts. Starch block cut into I c m s e g m e n t s a n d extracted. P r o t e i n (broken line) a n d R N A (solid line) c o n t e n t s of e x t r a c t s given as relative m o l a r quantities of m o n o m e r residues, based on residue weights of 116 a n d 345, respectively. A, I n t a c t nucleoprotein; B, Nucleoprotein in 7.7 M urea.

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Fig. 2. As in Fig. I. S o d i u m p h o s p h a t e , p H 8.2, ionic s t r e n g t h 0.08; 12 h at 15o volts. A, I n t a c t nucleoprotein; B, Nucleoprotein in 4 M urea; C, R N A (purchased; deproteinized and dialyzed). B o t h the urea t r e a t m e n t and t h e electrophoretic analyses were carried o u t at 0-3 °. The results were similar at urea concentrations of 4 M a n d 7-8 M, a n d those discussed in this note were o b t a i n e d f r o m electrophoretic r u n s b e g u n o. 5 to 3 h after addition of urea to the nucleop r o t e i n solution. The s t a r c h electrophoresis technique resembled t h a t described b y PARDEE el al. 9. Up to 4 r e c t a n g u l a r lucite t r o u g h s , 3o x 2 x I cm, were employed simultaneously at potentials of 300-450 or I5o volts. After the run, a filter p a p e r strip was pressed against the starch; R N A

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w a s located on t h e p a p e r b y m e a n s of a n u l t r a v i o l e t l a m p , a n d p r o t e i n b y a s t a i n i n g procedure. I n addition, t h e blocks were s o m e t i m e s c u t into s e g m e n t s a n d e x t r a c t e d o v e r n i g h t w i t h cold water, t h e R N A a n d p r o t e i n c o n t e n t s being e s t i m a t e d b y t h e u l t r a v i o l e t a b s o r p t i o n a n d t h e Folin reaction, respectively. As e m p l o y e d here, t h i s p r o c e d u r e w a s a t best s e m i - q u a n t i t a t i v e , since no a t t e m p t w a s m a d e to achieve c o m p l e t e e x t r a c t i o n of R N A a n d p r o t e i n f r o m t h e starch. Nevertheless, a coherent picture has emerged from these preliminary experiments. The electrophoretic findings m a y be s u m m a r i z e d as follows: I. The intact nucleoprotein (not e x p o s e d to urea). T h e n u c l e o p r o t e i n m i g r a t e d t o w a r d t h e a n o d e over t h e entire p H r a n g e f r o m 3.5-1o.6. A t p H 4 a n d lower, p r o t e i n a n d R N A m o v e d t o g e t h e r as a single e n t i t y . A t p H 5.5 a n d higher, t h e p a t t e r n i l l u s t r a t e d in Figs. I A a n d 2A w a s c o n s i s t e n t l y o b t a i n e d . T h i s p a t t e r n s h o w s a m i n o r p r o t e i n zone, a n d a faster zone c o n t a i n i n g b o t h R N A a n d p r o t e i n a n d p r e s u m a b l y nucleoprotein in character. I n t h i s p H r a n g e t h e distrib u t i o n s a n d mobilities were a p p a r e n t l y i n d e p e n d e n t of p H , since four blocks r u n s i m u l t a n e o u s l y at p H 6, 7, 8 a n d 9 g a v e identical p a t t e r n s . A t p H 7.7 t h e p a t t e r n was essentially u n c h a n g e d w h e n t h e ionic s t r e n g t h of t h e electrophoresis buffer w a s lowered f r o m o.I to 0.05. 2. The protein moiety alone. (Prepared b y mild alkaline h y d r o l y s i s of t h e n u c l e o p r o t e i n a n d r e m o v a l of t h e R N A h y d r o l y s i s p r o d u c t s b y dialysis.) M i g r a t i o n w a s t o w a r d t h e c a t h o d e at p H 5.5 a n d t o w a r d t h e a n o d e a t p H 6.8. 3. The nucleoprotein after urea treatment. E x p o s u r e to u r e a p r o d u c e d t w o electrophoretically d i s t i n c t " R N A " c o m p o n e n t s (Figs. I B a n d 2B). E v i d e n c e p r e s e n t e d s e p a r a t e l y 1° i n d i c a t e s t h a t t h e slow c o m p o n e n t did n o t c o m e directly f r o m t h e nucleoprotein, b u t w a s a p r o d u c t of t h e s e c o n d a r y d e g r a d a t i o n of t h e f a s t c o m p o n e n t . Therefore, only t h e fast R N A c o m p o n e n t will be considered here. A t p H 7.7 a n d h i g h e r (ionic s t r e n g t h o.i) t h e fast R N A c o m p o n e n t m i g r a t e d m o r e r a p i d l y t h a n t h e i n t a c t n u c l e o p r o t e i n a n d w i t h a speed e q u a l to t h a t of free R N A (Fig. 2). (A control e x p e r i m e n t s h o w e d u r e a n o t to c h a n g e t h e m o b i l i t y of free RNA.) U n d e r t h e s e conditions t h e s e p a r a t i o n of R N A a n d p r o t e i n w a s essentially complete. I t w a s i n c o m p l e t e a t p H 7.7 w h e n t h e ionic s t r e n g t h w a s lowered to 0.05, a n d at p H 5.5, ionic s t r e n g t h o.i. A t p H 4 a n d lower, p r o t e i n a n d R N A m i g r a t e d t o g e t h e r t o w a r d t h e anode. I n s u m m a r y , u r e a t r e a t m e n t p r o d u c e d a n electrophoretic b e h a v i o r similar to t h a t s h o w n b y m i x t u r e s of solutions of p r o t e i n a n d nucleic acid, w h e r e t h e t w o s u b s t a n c e s a r e linked only b y electrostatic forces a n d m i g r a t e i n d e p e n d e n t l y w h e n similarly c h a r g e d a n d a t h i g h e n o u g h ionic s t r e n g t h s T M . W h e n n o t e x p o s e d to urea, however, t h e p r o t e i n a n d R N A of t h e n u c l e o p r o t e i n m o v e d t o g e t h e r u n d e r c o n d i t i o n s w h e r e electrostatic forces w o u l d n o t be e x p e c t e d to hold t h e m together. I t s e e m s r e a s o n a b l e to c o n c l u d e t h a t t h e s e r i b o n u c l e o p r o t e i n s c o n t a i n R N A a n d p r o t e i n linked t o g e t h e r b y b o n d s o t h e r t h a n electrostatic bonds, t h a t t h e s e b o n d s are dissociated b y urea, a n d t h a t t h e y are, therefore, h y d r o g e n bonds. I t c a n n o t be said w h e t h e r o t h e r t y p e s of n o n electrostatic b o n d s are p r e s e n t u n t i l a m o r e detailed i n v e s t i g a t i o n h a s b e e n m a d e . DAVID ELSON

Biochemistry Section, The Weizmann Institute of Science, Rehovoth (Israel) 1 J. W . LITTLEFIELD, E. B. KELLER, J. GROSS AND P. C. ZAMECNIK, J. Biol. Chem., 217 (i 955) i i i. 2 E. CHARGAFF, in E. CHARGAFF ANn J. N. DAVIDSON, The Nucleic Acids, Vol. I, A c a d e m i c Press, Inc., N e w York, 1955. 2 D. ELSON AND E. CHARGAFF, Biochim. Biophys. Acta, 17 (1955) 367 . 4 E. CHARGAFF, D. ELSON AND H. T. SHIGEURA, Nature, 178 (1956) 682. s R. A. Cox, A. S. JONES, G. E. MARSH AND A. R. PEACOCKE, Biochim. Biophys. Acta, 21 (1956) 576 • 6 F.-C. CHAO AND H. K. SCHACHMAN, Arch. Biochem. Biophys., 61 (1956) 22o. 7 C. H. PARSONS, JR., Arch. Biochem. Biophys., 47 (1953) 76. 8 K. TAKATA AND S. OSAWA, Biochim. Biophys. Acta, 24 (1957) 207. 9 A. B. PARDEE, K. PAIGEN AND L. S. PRESTIDGE, Biochim. Biophys. Acta, 23 (1957) 162. 10 D. ELSON, Biochim. Biophys. Acta, 27 (1958) 216. 11 L. G. LONGSWORTH AND D. A. MAClNNES, J. Gen. Physiol., 25 (1942) 507 . 12 E. GOLDWASSER AND F. W. PUTNAM, J. Phys. ~ Coll. Chem., 54 (195o) 79. Received S e p t e m b e r 2oth, 1957