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Polyadenylic acid-directed binding of oligolysyl transfer RNA to ribosomes Inhibition by lysyl and deacylated transfer RNA
98
BIOCHIMICA ET BIOPHYSICA ACTA
BBA 96170
POLYADENYLIC ACID-DIRECTED BINDING OF OLIGOLYSYL T R A N S F E R RNA TO RIBOSOMES I N H I B I T I O N BY LYSYL AND DEACYLATED T R A N S F E R RNA
T O S H I M I C H I I K E M U R A AND H I D E O F U K U T O M E
Department o/ Physics, Faculty o/Science, Kyoto University, Kyoto (Japan) (Received December 6th, 1968)
SUMMARY
Poly A-directed binding of oligolysyl-tRNA to the ribosomes of Escherichia coli was inhibited by both lysyl-tRNA and deacylated tRNA. The inhibition by deacylated tRNA was much weaker than that by lysyl-tRNA. Addition of a supernatant enzyme fraction and GTP did not change the inhibitory effect of deacylated tRNA at equilibrium. However, when ribosomes that were pre-incubated with a large amount of deacylated tRNA were used, the rate of binding of oligolysyl-tRNA was found to increase on addition of the enzyme fraction and GTP. Code-directed binding of lysyl-tRNA and phenylalanyl-tRNA to the ribosomes was inhibited almost completely by deacylated tRNA, when added in large amount.
INTRODUCTION
During the process of protein synthesis, aminoacyl-tRNA is transformed into polypeptidyl-tRNA on ribosomes, but the nature of the binding of polypeptidyltRNA to ribosomes has not been well understood. Oligolysyl-tRNA which is isolated from the Escherichia coli polylysine-synthesizing system by phenol extraction binds to the poly A-ribosome complex and reacts with puromycin in the absence of GTP and supernatant enzymes 1-4. GOTTESMAN4 has shown that, in the absence of GTP and enzymes, a single lysine residue from lysyl-tRNA is incorporated into the oligolysine of oligolysyl-tRNA attached to the poly A-ribosome complex. By investigating the nature of the binding of oligolysyl-tRNA, we may be able to obtain some information about the binding of the normal polypeptidyl-tRNA-ribosome complex formed during protein synthesis. RYCHLIK2 reported that the oligolysyl-tRNA binding was inhibited by deacylated tRNA, and GOTTESMAN4 reported that this binding was inhibited also by lysyl-tRNA. In this report we compare the inhibitory effects of deacylated tRNA and lysyl-tRNA on oligolysyl-tRNA binding, in order to elucidate the relationship between the bindings of these three tRNA's to ribosomes. Furthermore, we investigate whether or not the relations are changed by addition of a supernatant enzyme fraction and GTP. Biochim. Biophys. Acta, 182 (1969) 98-io 4
OLIGOLYSYL-tRNA BINDING TO RIBOSOMES
99
MATERIALS AND METHODS Deacylated t R N A 5, the enzyme fraction 5, ribosomes well washed in o.5 M NH4C1 (ref. 6) and crude ribosomes 5 were prepared according to the methods reported previously, from the cells of E. coli H strain, harvested in the lcgarithmic phase. ElaClOligolysyl-tR~,TA was prepared b y incubating deacylated t R N A and L14Cllysine in an E. coli polylysine-synthesizing system. The incubation mixture, in a volume of 5 ml, contained: 40 mM Tris-HC1 (pH 7.6), 13 mM magnesium acetate, 60 mM ammonium acetate, 6 mM/~-mercaptoethanol, 0.5/,mole of GTP, 8/,moles of ATP, 0.8 mg of poly A, 7 mg of deacylated tRNA, 7 A2s0 n m units of the enzyme fraction, 5 ° A2e0 n m units of crude ribosomes and 4 ° nmoles of E14C~1ysine (247 ~C/#mole). After incubation for IO rain at 37 °, t R N A was de-proteinized b y phenol extractions and then precipitated twice by addition of ethanol. In order to deacylate contaminating E14CIlysyl-tRNA preferentially, the precipitate was dissolved in 5 ml of 0.3 M Tris-HC1 (pH 7.6) and incubated at 37 ° for 15 min. The t R N A was then precipitated by addition of ethanol, dissolved in water, dialysed against i mM potassium acetate buffer (pH 5-3) and stored at --20 °. Paper chromatographic analysis of the peptides released by alkaline treatment of this preparation was performed according to the method of TANAKA AND TERAOKA7. When the concentration of E~4Cllysine monomer amounted to more than 15 % of the total radioactivity, the above pro=edure to eliminate E14CIlysyl-tRNA was repeated. Aminoacyl-tRNA was prepared by the previously described method 6. The extent of the binding of [14C]oligolysyl-tRNA and [14Claminoacyl-tRNA to ribosomes was assayed by the nitrocellulose membrane filtration method described by NIRENBERG AND LEDERs. The reaction mixture contained: 60 mM Tris-acetic acid (pH 7.3), 15 mM magnesium acetate, IOO mM ammonium acetate and 6 mM fl-mercaptoethanol. To this were added washed ribosomes, polynucleotides for a template, E14Cloligolysyl-tRNA or I~4Claminoacyl-tRNA and other tRNA's in varying amounts, in a final volume of o.i ml.
RESULTS AND DISCUSSION The general characteristics of the binding reaction of oligolysyl-tRNA to the well-washed ribosomes were found to be similar to those of lysyl-tRNA, as reported by other workers °, when the requirements for Mg 2+ and poly A, as well as the time course, were examined. In all the experiments reported here, the amounts of poly A and C14CJoligolysyl-tRNA were sufficiently in excess, so that the extent of oligolysylt R N A binding was linearly proportional to the ribosome concentration.
Inhibition o/ oligolysyl-tRNA binding by lysyl-tRNA and deacylated tRNA The binding of oligolysysl-tRNA was found to be inhibited in the presence of added non-radioactive lysyl-tRNA or deacylated tRNA. Fig. I shows the extent of the inhibition as a function of the amount of added E12C~lysyl-tRNA ( O ) or deacylated t R N A (A). The extent of binding was measured here after incubation for 12 rain, when the saturation level had been reached. I t can be seen that the inhibition b y Biochim. Biophys. Acta, 182 (1969) 98-1o4
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Fig. I. I n h i b i t i o n of [14C]oligolysyl-tRNA binding b y deacylated t R N A or p2C]lysyl-tRNA. E a c h reaction m i x t u r e contained, in s t a n d a r d a s s a y buffer, 0.36 A~60nn, units of ribosomes, o. 3 A,60um units of oligolysyl-tRNA (58oo counts/min), the indicated a m o u n t s of deacylated t R N A (A) or [l*C]lysyl-tRNA ( O ) and 15/2g of poly A. The s y m b o l ( A ) represents the control e x p e r i m e n t in which 20/zg of poly U w a s employed instead of poly A, w i t h v a r y i n g a m o u n t s of deacylated t R N A . I n c u b a t i o n was carried o u t at 34 ° for i2 min. The s y m b o l (~]) and the broken line represent the results of the e x p e r i m e n t to examine the e x t e n t of inhibition b y deacylated t R N A , w i t h an i n c u b a t i o n time of 6 rain. The s y m b o l ( × ) r e p r e s e n t s the result obtained for the assay m i x t u r e which contained 0.092 A280nm units of e n z y m e fraction and 60 nmoles of GTP, in addition to the c o m p o n e n t s described above. The incubation was at 34 ° for 14 min. Fig. 2. The effect of deacylated t R N A on previously b o u n d [14C]oligolysyl-tRNA. E a c h reaction m i x t u r e , in a total volume of 80/zl, contained, in the s t a n d a r d assay buffer, 0.24 $ 2 6 0 nm u n i t s of [l*C]oligolysly-tRNA (4600 counts/min), 0.36 A,s0 nm units of ribosomes and 15/zg of poly A. After the reaction m i x t u r e s were pre-incubated for io min, 7 A 360nm units of deacylated t R N A which was dissolved in 20/~1 of the a s s a y buffer was added, either w i t h 0.074 A zs0 nm units of enzyme fraction and 6o nmoles of G T P ( O ) , or w i t h o u t t h e m (/x). After the addition of deacylated t R N A , the a m o u n t of b o u n d [14C]oligolysyl-tRNA was m e a s u r e d after incubation at 34 ° for the indicated times. To a n o t h e r series of pre-incubated mixtures, 4.8 A,60 nm units of [12C]lysyl-tRNA were added w i t h o u t the enzyme fraction and G T P ( A ) , or, alternatively, the assay buffer (2o#1) alone was added ( × ).
deacylated tRNA is considerably weaker than that b y lysyl-tRNA; even with 3o times as much deacylated tRNA as [14C]oligolysyl-tRNA, the extent of the binding is decreased only to 4 ° % of the uninhibited control. Similar results for the inhibition by deacylated t R N A were obtained using samples of the deacylated tRNA that were prepared by alkaline treatment of the lysyl-tRNA used above. As shown below, this weak inhibition of oligolysyl-tRNA binding by deacylated t R N A is in contrast to the case of the inhibition of [14C]lysyl-tRNA binding by deacylated tRNA, where the effect of deacylated t R N A is much more prominent. We shall discuss some models to explain the results of Fig. I in the last part of this report, considering the results of the experiment on the inhibition of lysyl-tRNA binding by deacylated tRNA. The extent of the inhibition of oligolysyl-tRNA binding by deacylated t R N A was found to be dependent on incubation time. When the binding was measured after a short incubation time of 6 min, when the binding of the uninhibited control was at 8 7 % of the equilibrium level, the inhibition by deacylated t R N A was more marked, as shown in Fig. I ([-]), than when measured after a sufficiently long incubation (12 min). This might suggest that a fraction of deacylated t R N A initially bound to the ribosomes is gradually replaced by oligolysyl-tRNA during prolonged Biochim. Biophys. Acta, 182 (1969) 98-1o 4
OLIGOLYSYL-tRNA BINDING TO RIBOSOMES
I0I
incubation. This process, if true, apparently does not go on indefinitely, since longer incubation periods (I8 and 25 rain) gave the same results as the result obtained at i2-min incubation.
E]/ects o/supernatant enzymes and G T P at the equilibrium state In the system of oligolysyl-tRNA binding to ribosomes, the extent of the binding was found to be hardly affected by addition of GTP and supernatant enzymes, in amounts chosen so as to be sufficient for lysine polymerization in the system of poly A-directed polylysine synthesis from lysyl-tRNA. A similar observation has been reported b y RYCHLIK2. This seems to suggest, though not conclusively, that peptidebond formation between oligolysyl-tRNA's does not take place even in the presence of enzymes and GTP, in contrast to lysyl-tRNA. Therefore, it seems that the effect of GTP and enzymes on the inhibition of oligolysyl-tRNA binding by deacylated t R N A can be studied without interference from the peptide-elongation reaction. We show in Fig. I, by means of the symbol ( × ), the results of the inhibition experiment performed in the presence of G T P and the enzyme fraction. It can be seen that oligolysyl-tRNA binding is inhibited b y deacylated t R N A in the same manner as the case without these additives. This suggests that, even in the presence of GTP and enzymes, deacylated t R N A can bind to ribosomes and the nature of the binding of oligolysyl-tRNA is not changed in such a way that oligolysyl-tRNA is translocated from the sites accessible to deacylated t R N A to other sites inaccessible to it. E//ects o/enzymes and G T P on kinetic properties o/ the exchange between oligolysyl and deacylated t R N A ' s on ribosomes The above experiments were essentially concerned with competition between t R N A ' s at the equilibrium state. In this section, the kinetic properties of t R N A binding to ribosomes are presented. Firstly, we investigated how the initially bound oligolysyl-tRNA was expelled from the ribosomes b y deacylated tRNA, in the presence or absence of GTP and enzymes. After [14C]oligolysyl-tRNA was pre-incubated with ribosomes and poly A, 30 times as much deacylated t R N A as [14C]oligolysyl-tRNA was added, either with ( O ) or without (A) GTP and the enzyme fraction, and the kinetics of replacement of bound oligolysyl-tRNA were measured (Fig. 2). It can be seen that the initially bound oligolysyl-tRNA was released from the ribosomes on addition of deacylated tRNA. The final level of this replacement was not changed by the addition of GTP and enzymes, but a small accelerating effect on the kinetics was perceptible. Addition of the buffer instead of deacylated t R N A ( × ) hardly affected the extent of the binding .We also show in Fig. 2 that, if LI~C]lysyl-tRNA was added instead of deacylated tRNA without GTP and enzymes, oligolysyl-tRNA was released more effectively than deacylated t R N A (A). Next, we examined how oligolysyl-tRNA could bind to the ribosomes which were complexed with deacylated tRNA. After IO min pre-incubation of the ribosomes with poly A and a large amount of deacylated tRNA, C14C]oligolysyl-tRNA was added, either with or without the enzyme fraction and GTP. The time course of E14C]oligolysyl-tRNA binding to these preincubated ribosomes is shown in Fig. 3 for both cases, with (A) and without (El) GTP and enzymes. For the control experiment we show the time course of oligolysyl-tRNA binding to the ribosomes which were pre-incubated without deacylated t R N A (O). In the case without the enzyme fracBiochim. Biophys. Acta, 182 (1969) 98-1o4
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Fig. 3. The effect of enzymes and G T P on the rate of [14C]oligolysyl-tRNA binding to ribosomes pre-incubated w i t h deaeylated t R N A . E a c h reaction mixture, in a total volume of 85 #1, contained, in the s t a n d a r d assay buffer, o. 3 A260nm units of ribosomes, 2.9 A2eonm units of deacylated t R N A and 15/~g of poly A. After io min p r e - i n c u b a t i o n at 34 °, o. 3 A 260 nm units of [14C1oligolysylt R N A (58oo counts/min, dissolved in 15/A of the assay buffer) were added, either with o.o74 A2so nm units of the enzyme fraction and 60 nmoles of G T P (A), or w i t b o u t t h e m (rq). The a m o u n t of b o u n d [14Cloligolysyl-tRNA w a s m e a s u r e d at the indicated times. I n the control series ( O ) , deacylated t R N A in the p r e - i n c u b a t i o n mixture, and the enzyme fraction and G T P in the reaction m i x t u r e were omitted. Fig. 4. Inhibition of [14C]lysyl-tRNA or [14C]phenylalanyl-tRNA binding to ribosomes b y deacylated t R N A . E a c h reaction mixture, in a total volume of o.I ml, contained, in the s t a n d a r d assay buffer, o.8 A2,0mn units of [t4C]lysyl-tRNA (47oo c o u n t s / m i n ) ((2))or o.8 A2e0n m units of [14C]phenylalanyl t R N A (44oo c o u n t s / m i n ) (A), 20 /~g of poly A or poly U, respectively, i . I A260n m units of ribosomes and the indicated a m o u n t s of deacylated t R N A . I n c u b a t i o n was at 34 ° for 13 min.
tion and GTP, not only was the final level of oligolysysl-tRNA binding lowered, but also the rate of binding was reduced markedly by the pre-incubation of the ribosomes with deacylated t R N A (D). This reduction of the rate of oligolysyl-tRNA binding m a y be due to a time-dependent displacement of the previously bound deacylated t R N A from the ribosomes. In the presence of GTP and the enzymes (A), the final level of oligolysyl-tRNA binding was also lowered to the same level as in the case without these additives, but the rate at which the final level was reached was accelerated to the same degree as would be expected if the ribosomes were not complexed to the deacylated tRNA. In separate experiments we observed that the addition of either the enzyme fraction or GTP alone had only a small effect on this rate and also that the rate of binding of oligolysyl-tRNA to the ribosomes which were not preincubated with deacylated tRNA was hardly affected by GTP and the enzymes in contrast to the case where the ribosomes were pre-incubated with deacylated tRNA. The data in Fig. 3 indicate that although the enzymes and GTP do not change the extent of inhibition by deacylated t R N A as far as it is seen at the equilibrium level, they enhance the rate of the displacement of the previously bound deacylated t R N A by oligolysyl-tRNA. In the process of protein synthesis, deacylated t R N A which might bind to the mRNA-ribosome complex will be unnecessary and inhibitory for peptide elongation. The above effects of enzymes and GTP will appear to be useful for the presumed process of eliminating bound deacylated t R N A from ribosomes. Biochim. Biophys. Acta, 182 (1969) 98-1o4
OLIGOLYSYL-tRNA BINDING TO RIBOSOMES
103
Inhibition o/ lysyl-tRNA binding by deacylated tRNA The results shown in Figs. I and 2 indicate that the inhibition of oligolysylt R N A binding by deacylated t R N A is weaker than that by lysyl-tRNA. A possible interpretation of this difference is that oligolysyl-tRNA can bind to more than one ribosomal site and lysyl-tRNA can bind to all of the sites, whereas deacylated t R N A does not have access to some of them. If this interpretation were true, the inhibition of lysyl-tRNA binding by deacylated tRNA should be partial even on addition of a large amount of deacylated tRNA. Experiments relevant to this question have been carried out by KURLAND1° and SEEDS et al. n on the poly U-directed binding of phenylalanyl-t RNA to ribosomes, but their results on the degree of inhibition by deacylated tRNA do not agree with each other. SEEDS et al. n reported that the extent of the binding was inhibited in a manner which suggested two binding sites for aminoacyltRNA, one sensitive to inhibition by deacylated tRNA and the other insensitive. On the other hand, the work of KURLAND1° was indicative of binding sites with equal affinity for deacylated and aminoacylated tRNA. Recently, LEVlN AND NIRENBERG12 reported that in the code-directed binding of deacylated t R N A and [14C~phenylalanyl-tRNA, the extent of the binding of deacylated phenylalanine t R N A was approximately equal to that of phenylalanyl-tRNA. We show in Fig. 4, the results of our experiments for the examination of the inhibition of the code-directed binding of I14CIlysy 1-tRNA ((~D) or I14Clphenylalanyl-tRNA (4) by deacylated tRNA. For both lysyl- and phenylalanyl-tRNA's, an overall decrease in binding is seen when a large amount of deacylated tRNA is added. This result is similar to that reported by KURLAND1° for the case of phenylalanyl-tRNA binding. Thus, our results, together with those of KURLAND1° and of LEVIN AND NIRENBERG12, seem to be contradictory to the above-mentioned interpretation for explanation of the difference between the inhibitory effects of deacylated tRNA and lysyl-tRNA on oligolysyl-tRNA binding as presented in Figs. I and 2. Another possible interpretation is that deacylated t R N A can bind to all sites accessible to oligolysyl-tRNA and lysyl-tRNA, but the stability of the deacylated tRNA-ribosome complex is much weaker than the stabilities of the lysyl-tRNA-ribosome and oligolysyl-tRNA-ribosome complexes. This interpretation is consistent with the observation, shown in Fig. I, that the extent of the inhibition of oligolysyl-tRNA binding by deacylated t R N A is larger at the short incubation time than at the long one. For, in the presence of a large excess of deacylated tRNA, compared with oligolysyl-tRNA, the ribosomes m a y have been occupied mainly by deacylated t R N A at first, but during the prolonged incubation replacement of deacylated t R N A b y oligolysyl-tRNA would occur in order to reach the equilibrium level, if the stability of the deacylated tRNA-ribosome complex were weaker to some extent than that of the oligolysyl-tRNA-ribosome complex. At present the second interpretation appears more plausible to us. However, further alternative models might be conceivable and further work is necessary in order to elucidate the exact mechanism that underlies the present observations. ACKNOWLEDGEMENT
The authors would like to express their sincere thanks to Dr. Y. Kawade of the Institute for Virus Research of Kyoto University for his valuable suggestions. This work was aided, in part, by a grant from the Ministry of Education. Biochim. Biophys. Acta, 182 (1969) 98-1o4
lO4
T. IKEMURA, H. FUKUTOME
REFERENCES I 2 3 4 5 6 7 8 9 io II 12
I. RYCHLIK, Biochim. Biophys. Acta, 114 (1965) 425 . I. RYCHLIK, Collection Czech. Chem. Commun., 31 (1966) 2583. I. H. GOLDBERG AND K. MITStI6I, Biochemistry, 6 (1967) 383 . M. E. GOTTESMAN, J. Biol. Chem., 242 (1967) 5564. H. KAGAWA, H. FUKUTOME AND Y. KAWADE, J. Mol. Biol., 26 (1967) 249. I. AOKI, T. IKEMURA, H. FUKUTOME AND Y. KAWADE, Biochim. Biophys. Acta, 179 (1969) 308. K. TANAKA AND H. TERAOKA, Biochim. Biophys. Acta, 114 (1966) 206. M. NIRENBERG AND P. LEDER, Science, 145 (1964) 1399. S. PESTKA AND M. NIRENBERG, Cold Spring Harbor Syrup. Quant. Biol., 31 (1966) 641. C. G. KtIRLAND, J. Mol. Biol., 18 (1966) 90. N. W. SEEDS, J. A. RETSEMA AND T. W. CONWAY, J. Mol. Biol., 27 (1967) 421. J. G. LEVlN AND M. NIRENBERG, J. Mol. Biol., 34 (1968) 467 .
Biochim. Biophys. Acta, 182 (1969) 98-1o4
BIOCHIMICA ET BIOPHYSICA ACTA
BBA 96170
POLYADENYLIC ACID-DIRECTED BINDING OF OLIGOLYSYL T R A N S F E R RNA TO RIBOSOMES I N H I B I T I O N BY LYSYL AND DEACYLATED T R A N S F E R RNA
T O S H I M I C H I I K E M U R A AND H I D E O F U K U T O M E
Department o/ Physics, Faculty o/Science, Kyoto University, Kyoto (Japan) (Received December 6th, 1968)
SUMMARY
Poly A-directed binding of oligolysyl-tRNA to the ribosomes of Escherichia coli was inhibited by both lysyl-tRNA and deacylated tRNA. The inhibition by deacylated tRNA was much weaker than that by lysyl-tRNA. Addition of a supernatant enzyme fraction and GTP did not change the inhibitory effect of deacylated tRNA at equilibrium. However, when ribosomes that were pre-incubated with a large amount of deacylated tRNA were used, the rate of binding of oligolysyl-tRNA was found to increase on addition of the enzyme fraction and GTP. Code-directed binding of lysyl-tRNA and phenylalanyl-tRNA to the ribosomes was inhibited almost completely by deacylated tRNA, when added in large amount.
INTRODUCTION
During the process of protein synthesis, aminoacyl-tRNA is transformed into polypeptidyl-tRNA on ribosomes, but the nature of the binding of polypeptidyltRNA to ribosomes has not been well understood. Oligolysyl-tRNA which is isolated from the Escherichia coli polylysine-synthesizing system by phenol extraction binds to the poly A-ribosome complex and reacts with puromycin in the absence of GTP and supernatant enzymes 1-4. GOTTESMAN4 has shown that, in the absence of GTP and enzymes, a single lysine residue from lysyl-tRNA is incorporated into the oligolysine of oligolysyl-tRNA attached to the poly A-ribosome complex. By investigating the nature of the binding of oligolysyl-tRNA, we may be able to obtain some information about the binding of the normal polypeptidyl-tRNA-ribosome complex formed during protein synthesis. RYCHLIK2 reported that the oligolysyl-tRNA binding was inhibited by deacylated tRNA, and GOTTESMAN4 reported that this binding was inhibited also by lysyl-tRNA. In this report we compare the inhibitory effects of deacylated tRNA and lysyl-tRNA on oligolysyl-tRNA binding, in order to elucidate the relationship between the bindings of these three tRNA's to ribosomes. Furthermore, we investigate whether or not the relations are changed by addition of a supernatant enzyme fraction and GTP. Biochim. Biophys. Acta, 182 (1969) 98-io 4
OLIGOLYSYL-tRNA BINDING TO RIBOSOMES
99
MATERIALS AND METHODS Deacylated t R N A 5, the enzyme fraction 5, ribosomes well washed in o.5 M NH4C1 (ref. 6) and crude ribosomes 5 were prepared according to the methods reported previously, from the cells of E. coli H strain, harvested in the lcgarithmic phase. ElaClOligolysyl-tR~,TA was prepared b y incubating deacylated t R N A and L14Cllysine in an E. coli polylysine-synthesizing system. The incubation mixture, in a volume of 5 ml, contained: 40 mM Tris-HC1 (pH 7.6), 13 mM magnesium acetate, 60 mM ammonium acetate, 6 mM/~-mercaptoethanol, 0.5/,mole of GTP, 8/,moles of ATP, 0.8 mg of poly A, 7 mg of deacylated tRNA, 7 A2s0 n m units of the enzyme fraction, 5 ° A2e0 n m units of crude ribosomes and 4 ° nmoles of E14C~1ysine (247 ~C/#mole). After incubation for IO rain at 37 °, t R N A was de-proteinized b y phenol extractions and then precipitated twice by addition of ethanol. In order to deacylate contaminating E14CIlysyl-tRNA preferentially, the precipitate was dissolved in 5 ml of 0.3 M Tris-HC1 (pH 7.6) and incubated at 37 ° for 15 min. The t R N A was then precipitated by addition of ethanol, dissolved in water, dialysed against i mM potassium acetate buffer (pH 5-3) and stored at --20 °. Paper chromatographic analysis of the peptides released by alkaline treatment of this preparation was performed according to the method of TANAKA AND TERAOKA7. When the concentration of E~4Cllysine monomer amounted to more than 15 % of the total radioactivity, the above pro=edure to eliminate E14CIlysyl-tRNA was repeated. Aminoacyl-tRNA was prepared by the previously described method 6. The extent of the binding of [14C]oligolysyl-tRNA and [14Claminoacyl-tRNA to ribosomes was assayed by the nitrocellulose membrane filtration method described by NIRENBERG AND LEDERs. The reaction mixture contained: 60 mM Tris-acetic acid (pH 7.3), 15 mM magnesium acetate, IOO mM ammonium acetate and 6 mM fl-mercaptoethanol. To this were added washed ribosomes, polynucleotides for a template, E14Cloligolysyl-tRNA or I~4Claminoacyl-tRNA and other tRNA's in varying amounts, in a final volume of o.i ml.
RESULTS AND DISCUSSION The general characteristics of the binding reaction of oligolysyl-tRNA to the well-washed ribosomes were found to be similar to those of lysyl-tRNA, as reported by other workers °, when the requirements for Mg 2+ and poly A, as well as the time course, were examined. In all the experiments reported here, the amounts of poly A and C14CJoligolysyl-tRNA were sufficiently in excess, so that the extent of oligolysylt R N A binding was linearly proportional to the ribosome concentration.
Inhibition o/ oligolysyl-tRNA binding by lysyl-tRNA and deacylated tRNA The binding of oligolysysl-tRNA was found to be inhibited in the presence of added non-radioactive lysyl-tRNA or deacylated tRNA. Fig. I shows the extent of the inhibition as a function of the amount of added E12C~lysyl-tRNA ( O ) or deacylated t R N A (A). The extent of binding was measured here after incubation for 12 rain, when the saturation level had been reached. I t can be seen that the inhibition b y Biochim. Biophys. Acta, 182 (1969) 98-1o4
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Fig. I. I n h i b i t i o n of [14C]oligolysyl-tRNA binding b y deacylated t R N A or p2C]lysyl-tRNA. E a c h reaction m i x t u r e contained, in s t a n d a r d a s s a y buffer, 0.36 A~60nn, units of ribosomes, o. 3 A,60um units of oligolysyl-tRNA (58oo counts/min), the indicated a m o u n t s of deacylated t R N A (A) or [l*C]lysyl-tRNA ( O ) and 15/2g of poly A. The s y m b o l ( A ) represents the control e x p e r i m e n t in which 20/zg of poly U w a s employed instead of poly A, w i t h v a r y i n g a m o u n t s of deacylated t R N A . I n c u b a t i o n was carried o u t at 34 ° for i2 min. The s y m b o l (~]) and the broken line represent the results of the e x p e r i m e n t to examine the e x t e n t of inhibition b y deacylated t R N A , w i t h an i n c u b a t i o n time of 6 rain. The s y m b o l ( × ) r e p r e s e n t s the result obtained for the assay m i x t u r e which contained 0.092 A280nm units of e n z y m e fraction and 60 nmoles of GTP, in addition to the c o m p o n e n t s described above. The incubation was at 34 ° for 14 min. Fig. 2. The effect of deacylated t R N A on previously b o u n d [14C]oligolysyl-tRNA. E a c h reaction m i x t u r e , in a total volume of 80/zl, contained, in the s t a n d a r d assay buffer, 0.24 $ 2 6 0 nm u n i t s of [l*C]oligolysly-tRNA (4600 counts/min), 0.36 A,s0 nm units of ribosomes and 15/zg of poly A. After the reaction m i x t u r e s were pre-incubated for io min, 7 A 360nm units of deacylated t R N A which was dissolved in 20/~1 of the a s s a y buffer was added, either w i t h 0.074 A zs0 nm units of enzyme fraction and 6o nmoles of G T P ( O ) , or w i t h o u t t h e m (/x). After the addition of deacylated t R N A , the a m o u n t of b o u n d [14C]oligolysyl-tRNA was m e a s u r e d after incubation at 34 ° for the indicated times. To a n o t h e r series of pre-incubated mixtures, 4.8 A,60 nm units of [12C]lysyl-tRNA were added w i t h o u t the enzyme fraction and G T P ( A ) , or, alternatively, the assay buffer (2o#1) alone was added ( × ).
deacylated tRNA is considerably weaker than that b y lysyl-tRNA; even with 3o times as much deacylated tRNA as [14C]oligolysyl-tRNA, the extent of the binding is decreased only to 4 ° % of the uninhibited control. Similar results for the inhibition by deacylated t R N A were obtained using samples of the deacylated tRNA that were prepared by alkaline treatment of the lysyl-tRNA used above. As shown below, this weak inhibition of oligolysyl-tRNA binding by deacylated t R N A is in contrast to the case of the inhibition of [14C]lysyl-tRNA binding by deacylated tRNA, where the effect of deacylated t R N A is much more prominent. We shall discuss some models to explain the results of Fig. I in the last part of this report, considering the results of the experiment on the inhibition of lysyl-tRNA binding by deacylated tRNA. The extent of the inhibition of oligolysyl-tRNA binding by deacylated t R N A was found to be dependent on incubation time. When the binding was measured after a short incubation time of 6 min, when the binding of the uninhibited control was at 8 7 % of the equilibrium level, the inhibition by deacylated t R N A was more marked, as shown in Fig. I ([-]), than when measured after a sufficiently long incubation (12 min). This might suggest that a fraction of deacylated t R N A initially bound to the ribosomes is gradually replaced by oligolysyl-tRNA during prolonged Biochim. Biophys. Acta, 182 (1969) 98-1o 4
OLIGOLYSYL-tRNA BINDING TO RIBOSOMES
I0I
incubation. This process, if true, apparently does not go on indefinitely, since longer incubation periods (I8 and 25 rain) gave the same results as the result obtained at i2-min incubation.
E]/ects o/supernatant enzymes and G T P at the equilibrium state In the system of oligolysyl-tRNA binding to ribosomes, the extent of the binding was found to be hardly affected by addition of GTP and supernatant enzymes, in amounts chosen so as to be sufficient for lysine polymerization in the system of poly A-directed polylysine synthesis from lysyl-tRNA. A similar observation has been reported b y RYCHLIK2. This seems to suggest, though not conclusively, that peptidebond formation between oligolysyl-tRNA's does not take place even in the presence of enzymes and GTP, in contrast to lysyl-tRNA. Therefore, it seems that the effect of GTP and enzymes on the inhibition of oligolysyl-tRNA binding by deacylated t R N A can be studied without interference from the peptide-elongation reaction. We show in Fig. I, by means of the symbol ( × ), the results of the inhibition experiment performed in the presence of G T P and the enzyme fraction. It can be seen that oligolysyl-tRNA binding is inhibited b y deacylated t R N A in the same manner as the case without these additives. This suggests that, even in the presence of GTP and enzymes, deacylated t R N A can bind to ribosomes and the nature of the binding of oligolysyl-tRNA is not changed in such a way that oligolysyl-tRNA is translocated from the sites accessible to deacylated t R N A to other sites inaccessible to it. E//ects o/enzymes and G T P on kinetic properties o/ the exchange between oligolysyl and deacylated t R N A ' s on ribosomes The above experiments were essentially concerned with competition between t R N A ' s at the equilibrium state. In this section, the kinetic properties of t R N A binding to ribosomes are presented. Firstly, we investigated how the initially bound oligolysyl-tRNA was expelled from the ribosomes b y deacylated tRNA, in the presence or absence of GTP and enzymes. After [14C]oligolysyl-tRNA was pre-incubated with ribosomes and poly A, 30 times as much deacylated t R N A as [14C]oligolysyl-tRNA was added, either with ( O ) or without (A) GTP and the enzyme fraction, and the kinetics of replacement of bound oligolysyl-tRNA were measured (Fig. 2). It can be seen that the initially bound oligolysyl-tRNA was released from the ribosomes on addition of deacylated tRNA. The final level of this replacement was not changed by the addition of GTP and enzymes, but a small accelerating effect on the kinetics was perceptible. Addition of the buffer instead of deacylated t R N A ( × ) hardly affected the extent of the binding .We also show in Fig. 2 that, if LI~C]lysyl-tRNA was added instead of deacylated tRNA without GTP and enzymes, oligolysyl-tRNA was released more effectively than deacylated t R N A (A). Next, we examined how oligolysyl-tRNA could bind to the ribosomes which were complexed with deacylated tRNA. After IO min pre-incubation of the ribosomes with poly A and a large amount of deacylated tRNA, C14C]oligolysyl-tRNA was added, either with or without the enzyme fraction and GTP. The time course of E14C]oligolysyl-tRNA binding to these preincubated ribosomes is shown in Fig. 3 for both cases, with (A) and without (El) GTP and enzymes. For the control experiment we show the time course of oligolysyl-tRNA binding to the ribosomes which were pre-incubated without deacylated t R N A (O). In the case without the enzyme fracBiochim. Biophys. Acta, 182 (1969) 98-1o4
102
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Fig. 3. The effect of enzymes and G T P on the rate of [14C]oligolysyl-tRNA binding to ribosomes pre-incubated w i t h deaeylated t R N A . E a c h reaction mixture, in a total volume of 85 #1, contained, in the s t a n d a r d assay buffer, o. 3 A260nm units of ribosomes, 2.9 A2eonm units of deacylated t R N A and 15/~g of poly A. After io min p r e - i n c u b a t i o n at 34 °, o. 3 A 260 nm units of [14C1oligolysylt R N A (58oo counts/min, dissolved in 15/A of the assay buffer) were added, either with o.o74 A2so nm units of the enzyme fraction and 60 nmoles of G T P (A), or w i t b o u t t h e m (rq). The a m o u n t of b o u n d [14Cloligolysyl-tRNA w a s m e a s u r e d at the indicated times. I n the control series ( O ) , deacylated t R N A in the p r e - i n c u b a t i o n mixture, and the enzyme fraction and G T P in the reaction m i x t u r e were omitted. Fig. 4. Inhibition of [14C]lysyl-tRNA or [14C]phenylalanyl-tRNA binding to ribosomes b y deacylated t R N A . E a c h reaction mixture, in a total volume of o.I ml, contained, in the s t a n d a r d assay buffer, o.8 A2,0mn units of [t4C]lysyl-tRNA (47oo c o u n t s / m i n ) ((2))or o.8 A2e0n m units of [14C]phenylalanyl t R N A (44oo c o u n t s / m i n ) (A), 20 /~g of poly A or poly U, respectively, i . I A260n m units of ribosomes and the indicated a m o u n t s of deacylated t R N A . I n c u b a t i o n was at 34 ° for 13 min.
tion and GTP, not only was the final level of oligolysysl-tRNA binding lowered, but also the rate of binding was reduced markedly by the pre-incubation of the ribosomes with deacylated t R N A (D). This reduction of the rate of oligolysyl-tRNA binding m a y be due to a time-dependent displacement of the previously bound deacylated t R N A from the ribosomes. In the presence of GTP and the enzymes (A), the final level of oligolysyl-tRNA binding was also lowered to the same level as in the case without these additives, but the rate at which the final level was reached was accelerated to the same degree as would be expected if the ribosomes were not complexed to the deacylated tRNA. In separate experiments we observed that the addition of either the enzyme fraction or GTP alone had only a small effect on this rate and also that the rate of binding of oligolysyl-tRNA to the ribosomes which were not preincubated with deacylated tRNA was hardly affected by GTP and the enzymes in contrast to the case where the ribosomes were pre-incubated with deacylated tRNA. The data in Fig. 3 indicate that although the enzymes and GTP do not change the extent of inhibition by deacylated t R N A as far as it is seen at the equilibrium level, they enhance the rate of the displacement of the previously bound deacylated t R N A by oligolysyl-tRNA. In the process of protein synthesis, deacylated t R N A which might bind to the mRNA-ribosome complex will be unnecessary and inhibitory for peptide elongation. The above effects of enzymes and GTP will appear to be useful for the presumed process of eliminating bound deacylated t R N A from ribosomes. Biochim. Biophys. Acta, 182 (1969) 98-1o4
OLIGOLYSYL-tRNA BINDING TO RIBOSOMES
103
Inhibition o/ lysyl-tRNA binding by deacylated tRNA The results shown in Figs. I and 2 indicate that the inhibition of oligolysylt R N A binding by deacylated t R N A is weaker than that by lysyl-tRNA. A possible interpretation of this difference is that oligolysyl-tRNA can bind to more than one ribosomal site and lysyl-tRNA can bind to all of the sites, whereas deacylated t R N A does not have access to some of them. If this interpretation were true, the inhibition of lysyl-tRNA binding by deacylated tRNA should be partial even on addition of a large amount of deacylated tRNA. Experiments relevant to this question have been carried out by KURLAND1° and SEEDS et al. n on the poly U-directed binding of phenylalanyl-t RNA to ribosomes, but their results on the degree of inhibition by deacylated tRNA do not agree with each other. SEEDS et al. n reported that the extent of the binding was inhibited in a manner which suggested two binding sites for aminoacyltRNA, one sensitive to inhibition by deacylated tRNA and the other insensitive. On the other hand, the work of KURLAND1° was indicative of binding sites with equal affinity for deacylated and aminoacylated tRNA. Recently, LEVlN AND NIRENBERG12 reported that in the code-directed binding of deacylated t R N A and [14C~phenylalanyl-tRNA, the extent of the binding of deacylated phenylalanine t R N A was approximately equal to that of phenylalanyl-tRNA. We show in Fig. 4, the results of our experiments for the examination of the inhibition of the code-directed binding of I14CIlysy 1-tRNA ((~D) or I14Clphenylalanyl-tRNA (4) by deacylated tRNA. For both lysyl- and phenylalanyl-tRNA's, an overall decrease in binding is seen when a large amount of deacylated tRNA is added. This result is similar to that reported by KURLAND1° for the case of phenylalanyl-tRNA binding. Thus, our results, together with those of KURLAND1° and of LEVIN AND NIRENBERG12, seem to be contradictory to the above-mentioned interpretation for explanation of the difference between the inhibitory effects of deacylated tRNA and lysyl-tRNA on oligolysyl-tRNA binding as presented in Figs. I and 2. Another possible interpretation is that deacylated t R N A can bind to all sites accessible to oligolysyl-tRNA and lysyl-tRNA, but the stability of the deacylated tRNA-ribosome complex is much weaker than the stabilities of the lysyl-tRNA-ribosome and oligolysyl-tRNA-ribosome complexes. This interpretation is consistent with the observation, shown in Fig. I, that the extent of the inhibition of oligolysyl-tRNA binding by deacylated t R N A is larger at the short incubation time than at the long one. For, in the presence of a large excess of deacylated tRNA, compared with oligolysyl-tRNA, the ribosomes m a y have been occupied mainly by deacylated t R N A at first, but during the prolonged incubation replacement of deacylated t R N A b y oligolysyl-tRNA would occur in order to reach the equilibrium level, if the stability of the deacylated tRNA-ribosome complex were weaker to some extent than that of the oligolysyl-tRNA-ribosome complex. At present the second interpretation appears more plausible to us. However, further alternative models might be conceivable and further work is necessary in order to elucidate the exact mechanism that underlies the present observations. ACKNOWLEDGEMENT
The authors would like to express their sincere thanks to Dr. Y. Kawade of the Institute for Virus Research of Kyoto University for his valuable suggestions. This work was aided, in part, by a grant from the Ministry of Education. Biochim. Biophys. Acta, 182 (1969) 98-1o4
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T. IKEMURA, H. FUKUTOME
REFERENCES I 2 3 4 5 6 7 8 9 io II 12
I. RYCHLIK, Biochim. Biophys. Acta, 114 (1965) 425 . I. RYCHLIK, Collection Czech. Chem. Commun., 31 (1966) 2583. I. H. GOLDBERG AND K. MITStI6I, Biochemistry, 6 (1967) 383 . M. E. GOTTESMAN, J. Biol. Chem., 242 (1967) 5564. H. KAGAWA, H. FUKUTOME AND Y. KAWADE, J. Mol. Biol., 26 (1967) 249. I. AOKI, T. IKEMURA, H. FUKUTOME AND Y. KAWADE, Biochim. Biophys. Acta, 179 (1969) 308. K. TANAKA AND H. TERAOKA, Biochim. Biophys. Acta, 114 (1966) 206. M. NIRENBERG AND P. LEDER, Science, 145 (1964) 1399. S. PESTKA AND M. NIRENBERG, Cold Spring Harbor Syrup. Quant. Biol., 31 (1966) 641. C. G. KtIRLAND, J. Mol. Biol., 18 (1966) 90. N. W. SEEDS, J. A. RETSEMA AND T. W. CONWAY, J. Mol. Biol., 27 (1967) 421. J. G. LEVlN AND M. NIRENBERG, J. Mol. Biol., 34 (1968) 467 .
Biochim. Biophys. Acta, 182 (1969) 98-1o4