Guanosine 5-0-(3-Thiotriphosphate) as an Analog of GTP in Protein Biosynthesis

Transcription

1 THE JOURNAL OF BIOLOGICAL CHEMISTRY by The American Society of Biological Chemists, Inc. Vol. 261, No. 7, Issue of March 5, pp ,1986 Printed in U.S.A. Guanosine 5-0-(3-Thiotriphosphate) as an Analog of GTP in Protein Biosynthesis THE EFFECTS OF TEMPERATURE AND POLYCATIONS ON THE ACCURACY OF INITIAL RECOGNITION OF AMINOACYL-tRNA TERNARY COMPLEXES BY RIBOSOMES* (Received for publication, May 10, 1985) Amr M. Karim+ and Robert C. Thompson From the Department of Molecular, Cellular, and Developmental Biology, University of Colorado at Boulder, Boulder, Colorado Guanosine 5-0-(3-thio)triphosphate (GTP-yS) is a good analog of GTP in the reactions leading to the formation of a peptide bond in protein biosynthesis. It forms binary and ternary complexes with elongation factor Tu (EF-Tu), and with EF-Tu and In addition, it stimulates aa-trna binding to ribosomes. Although GTP-yS hydrolysis is more than three orders of magnitude slower GTP than hydrolysis, both reactions are dependent on the formationofanoncovalentcomplex (RS-TC) between mrna-programmed ribosomes and ternary complex, and the complexes resulting from that hydrolysis are intermediates in peptide formation. (5,6). The rate constants for initial recognition of the ternary complex have been more difficult to determine, primarily because the hydrolysis reaction is so fast. To circumvent this The rate of dissociation of the ribosome-ef- problem, we have studied the analogous reaction in which Tu*GTP-yS-aa-tRNAcomplex was determinedfrom GTP is replaced by GTPyS. The first part of this paper the rate of labeled peptide formation in the presence reports of some of the properties of the ternary complex conan unlabeled ternary complex chase. This rate (2.2 X taining GTPyS and describes its interaction with the mrnas-l) is similar to that determined previously programmed ribosome. The results support the view that the (Thompson, R. C., and Karim, A. M. (1982) Proc. Nutl. GTPyS ternary complex is closely analogous to the ternary Acad. Sei. U. S. A. 79, ) from the progress complex containing GTP, and that some of the rate constants of GTP-yS hydrolysis. The effects of temperature and for reaction of the GTPyS complexes with the ribosome apply polycation concentration on this rate constant and that for GTP-yS hydrolysis are reported. The rate constants measured are consistent with a kinetic rather than thermodynamic limit on the accuracy of the aa-trna selection in vivo. The codon-anticodon based selection of aa-trnasl by mrna-programmed ribosomes is several orders of magnitude more accurate than base pairing in solution (1-3). This led Hopfield (4) to propose a mechanism, kinetic proofreading, by which the accuracy of aa-trna selection could be enhanced before the amino acid was incorporated into protein. We have presented experimental evidence in favor of this two-step selection of aa-trnas by showing that poly(u)- programmed ribosomes will hydrolyze the GTP of both cognate and near-cognate aa-trna-ef-tu. GTP complexes, although they bind aa-trna efficiently only from the former * This work was supported by Grant GM from the National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked aduertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 4 Present address: Biochemistry Dept., Faculty of Science, Ain Shams University, Cairo, Egypt. The abbreviations used are: aa-trna, aminoacyl-trna EF-Tu, elongation factor Tu; GTPrS, guanosine 5-0-(3-thiotriphosphate); RS. TC, ribosome. txnary complex; Ac, acetyl; GMPPNP, guanosine 5 -[P,y-imido]triphosphate complex. The aa-trna from near-cognate complexes is usually rejected following GTP hydrolysis (2, 3). We have elucidated some aspects of the mechanism of aatrna selection by measuring the rate constants for initial selection of ternary complexes by ribosomes and for proofreading of their aa-trna. The rate constants for acceptance and rejection of aa-trnas during proofreading have been determined through measuring the rate of peptide bond formation and the ratio of GTP hydrolyzed to peptide formed also to the ternary complex containing GTP. The results of our earlier studies (7) indicated that the accuracy of ternary complex selection is much higher with GTPyS than with GTP complexes. We also found that the dissociation of cognate ternary complexes from the ribosome is 10,000-fold slower than the rate of GTP hydrolysis. These observations led us to the conclusion that the accuracy of ternary complex selection by ribosomes is limited by the kinetics of reaction. We proposed that the ability of the ribosome to preferentially reject near-cognate ternary complexes is limited by the time available before GTP hydrolysis occurs, rather than any intrinsic inability of the ribosome to distinguish cognate from near-cognate ternary complexes. These studies proved that the ribosome has a much greater accuracy than it manifests in protein biosynthesis. However, the hypothesis that the speed of protein synthesis is the primary factor limiting its accuracy is less conclusive. The rate of cognate ternary complex dissociation from the ribosome need not be as low as times the rate of GTP hydrolysis to permit rapid use of this complex in protein synthesis. Either something other than the need for speed is the primary factor limiting the rate of ternary complex dissociation and the accuracy of ternary complex selection, or the rate of ternary complex dissociation is artificially slowed by the nonphysiological conditions used in the experiments. We have examined the latter possibility by measuring the rate constants for GTPyS hydrolysis and dissociation of the RS. TC at physiological temperatures and at low concentrations of polycations. Under these conditions the rate of

2 Accuracy of Ternary Complex Selection by Ribosomes 3239 GTPyS hydrolysis increases 10-fold but the rate of complex dissociation increases 1000-fold. When these changes are extrapolated to the physiological reaction with GTP they indicate that the rate of GTP hydrolysis is about 10-fold greater than the rate of RS.TC dissociation. This result reinforces the conclusion that accuracy is kinetically rather than thermodynamically limited and provides further evidence that the need for rapid protein synthesis is the primary factor dictating the less than maximal accuracy observed. 15 I MATERIALS AND METHODS Ribosomes were extracted from Escherichia coli, washed once with 1 M NH&l, and pelleted through a sucrose gradient as described previously (3). They were programmed by incubation for 10 min at 37 "C with 1.2 pg of poly(u) and 12 pmol of AC[~H]P~~-~RNA~~/A~~ 1 I I - c unit of ribosomes. Up to 520 A, units/ml of ribosomes were incu- 0 IO bated in a buffer consisting of 50 mm Tris. HCl (ph 7.2), 50 mm KC1, 10 mm MgClz, 1 mm dithiothreitol. Generally 30 to 40% of the ribosomes bound AcPhe-tRNAPhe as judged by a nitrocellulose filter assay (8). EF-Tu.GDP was a gift of Dr. J. F. Eccleston, National Institute for Medical Research, Mill Hill, London, United Kingdom. The GDP was removed by treatment with charcoal and 10 mm EDTA (3). trnaphe was purchased from Boehringer Mannheim and charged with [3H]Phe to about 1000 pmol/az6,,. [3H]Phe was purchased from Amersham Corp. at about 50 Ci/mmol. Ac[~H]P~~-~RNA'" was prepared as described by Lapidot et ul. (9). The specific activity of the [3H]Phe used in preparing Ac[~H]P~~-~RNA'~* was diluted to approximately 1 Ci/mmol before charging the trna. [35S]GTP-yS was either purchased from Amersham Corp. at a specific activity of approximately 30 Ci/mmol or prepared as described by Cassidy and Kerrick (10) from [35S]Na3P03S(1 Ci/mmol) supplied by New England Nuclear. Unless otherwise stated, ternary complexes were prepared by incubating GTPyS with EF-Tu and aa-trna at a ratio of 1:1.5:1.5 and a nucleotide concentration of 3 to 4 pm. Incubation was for 15 min in a buffer consisting of 50 mm Tris.HC1 (ph 7.2), 50 mm KC1, 5 mm MgClZ, and 1 mm dithiotreitol. The ternary complex was then centrifuged through a small Bio-Gel P-6 column prepared in a 0.5-ml syringe and equilibrated with the same buffer, or for experiments listed in Table 111, with a buffer that has the ionic composition described in Table 111. The Bio-Gel step was necessary in order to remove a background of 35S counts (approximately 7%) that were not adsorbed to charcoal. Nucleotide recovery from the Bio-Gel was usually 50 to 60%, in about the same volume that was added. Analysis for [35S]GTP-yS hydrolysis was performed on samples (up to 60 pl) that were quenched either in 100 p1 of 0.5 M KHzP04 or 4 volumes of 0.1 M EDTA and incubated for 5 min at 0 "C with 1 ml of a 5% suspension of charcoal in 50 mm KHzP04 and 50 mm Na3P03S to absorb unchanged [35S]GTP-yS. Binding of [3H]Phe-tRNAPhe to ribosomes was assayed by the nitrocellulose filter assay of Nirenberg and Leder (8). Samples were diluted into 1 ml of the filtration buffer (50 mm Tris.HC1, ph 7.2, 60 mm NH4Cl, and 5 mmmgc1,) and rapidly ( 4 s) filtered through nitrocellulose (Millipore HAWP). Analysis for peptide formation was performed on EDTA quenched samples by hydrolysis of the reaction mixture in 0.5 M aqueous Na2C0, acidification with 1 M aqueous HCl to a final ph of 1.0, and subsequent extraction of the labeled peptide (AcPhePheOH) with ethyl acetate. RESULTS csssl GTPYS (/AM) FIG. 1. Formation of an EF-Tu-GTP-yS binary complex. Increasing concentrations of [%]GTP-yS were incubated at 0 "C for 30 min at 5 mm M$+ in the absence (X) of EF-Tu or in the presence of 1 PM (0) and 5 ~ L M (0) EF-Tu. Ten-microliter samples were then diluted to about 2 ml with filtration buffer and rapidly filtered through nitrocellulose filters. The filters were then washed with about 3 ml of buffer. 50 p~ did not further increase the apparent efficiency of complex formation. For reactions at 5 p~ EF-Tu and 0.3 to 10 p~ [35S]GTPyS, the efficiency of complex formation as judged by 35S retention on the filters remained constant at 25 f 4% whether the limiting reactant was EF-Tu or [35S] GTPrS. The results described above suggest that binary complex formation is complete and that the low yield of 35S retained on nitrocellulose filters is due to the formation of a labile complex which dissociates during nitrocellulose filtration. This has been confirmed by the results of an experiment in which a 3-pl reaction mixture containing GTPyS (5 PM) and EF-Tu (1 p ~ was ) diluted 100-fold and incubated for 0.5 min before filtration; only f 0.02 pmol of GTPyS was retained compared to the 0.53 f 0.09 pmol retained for an identical reaction mixture that was diluted and filtered in less than 5 s. The binary complex formed between EF-Tu and GTPyS is similar to the analogous complex formed between EF-Tu and GTP in that it forms a ternary complex with aa-trna. By analogy with the GTP complex, conversion of an EF-Tu- GTPyS binary complex to an EF-Tu. GTP7 S. aa-trna ternary complex should be accompanied by the failure of radioactivity labeled GTPyS incorporated in the complex to bind to nitrocellulose filters (12). In accord with this expectation, a reduction in the amount of 35S bound to nitrocellulose filters was observed on addition of Phe-tRNA"' to a reaction mixture containing EF-Tu and [35S]GTPyS (Table I). EF-Tu.GTPyS WillStimulatetheBinding of the PhetRNAph to Poly(U)-programmed Ribosomes-The ternary complex formed between EF-Tu, GTPyS, and Phe-tRNAPh" is similar to the physiological complex containing GTP in that it stimulates the binding of Phe-tRNAPhe to poly(u)- programmed ribosomes. This is shown by the results of an experiment, in which [35S]GTPyS was added to a solution GTPyS as an Analog of GTP in Protein Biosynthesis GTPyS Can Replace GTP in Its Interaction with Phe-tRNA and EF-Tu-Like GTP (ll), GTPyS will form a nitrocellulose-filterable complex when incubated with EF-Tu. This is shown (Fig. 1) by the retention of 35S on nitrocellulose filters containbg EF-Tu, [3H]Phe-tRNAPhe, and poly(u)-proafter a 30-min 0 "C incubation of [35S]GTPyS with 1 or 5 p~ grammed ribosomes, and ribosome-bound [3H]Phe-tRNA was EF-Tu. For reactions at 1 p~ EF-Tu, the amount of 35S measured by its retention on nitrocellulose filters following a retained increased as the concentration of [35S]GTPyS was 30-s incubation at 0 "C. In the absence of GTPyS, very little raised from 0.3 to 10 p ~ At. 10 p~ [35S]GTPyS, the amount 3H was bound to the filter, but the addition of GTPyS resulted of 35S retained was equivalent to 20% of the amount of EF- Tu present and raising the concentration of [35S]GTPyS to in 3H binding. The molar ratio of GTPyS added to Phe-tRNA bound to the ribosomes was about 2. This departure from the

3 3240 Accuracy of Ternary Complex Ribosomes Selection by TABLE I Formation of binary complex between EF-Tu and GTPyS and ternary complex between EF-Tu, GTPyS, and Phe-tRNAPh Binary complex was prepared by mixing 630 pmol of [35S]GTPyS with 210 pmol of EF-Tu in 70 pl of reaction buffer (50 mm Tris-HC1, ph 7.2, 50 mm KCl, 5 mm MgCl,, and 1 mm dithiothreitol. Aliquots (9.6 pl) were diluted to 12 pl with reaction buffer containing 0 to 107 pmol of [3H]Phe-tRNAPhe and after a 15-min incubation at 0 C were diluted to 1 ml with filtration buffer (50 mm Tris.HC1, ph 7.2, 60 mm NHICl, and 5 mm MgCl,) and filtered through nitrocellulose. Addition [35S]GTPyS bound to filter pmol None 0.03 EF-TU (2.4 pm) 2.4 f 0.2 EF-Tu (2.4 p ~ ) Phe-tRNAPhe (0.55 p ~ ) 1.8 EF-Tu (2.4 p ~ ) Phe-tRNAPhe (1.1 p ~ ) EF-Tu (2.4 p ~ ) Phe-tRNAPhe (2.2 p ~ ) EF-Tu (2.4 p ~ ) Phe-tRNAPhe (4.4 p ~ ) 0.12 f 0.05 EF-Tu (2.4 p ~ + ) Phe-tRNAPhe (8.8 p ~ ) 0.11 f 0.04 expected 1:l stoichiometry is probably due to radioactive impurities in the GTPyS because, when the ternary complex was purified of low molecular weight contaminants on a Bio- Gel P-6 column (see "Materials and Methods") and then mixed with ribosomes, the ratio of ribosome-bound Phe-tRNA to GTPyS present was 1:1.25, approaching the ratio of 1:l expected for the ternary complex. In the course of experiments involving nitrocellulose filtration of the complex formed between poly(u)-programmed ribosomes and a [3H]Phe-tRNAPhe.EF-Tu. [35S]GTPyS ternary complex, we always observed that much less [3sS]GTPyS than [3H]Phe-tRNA was bound to the filters, even though little GTPyS hydrolysis had taken place at the time those samples were taken. In order to determine whether the low yield of filter-bound 3sS was due to GTPyS hydrolysis during the nitrocellulose filtration or to release of nucleotide from the RS. TC complex, we determined the chemical nature of the 35S found in the filtrate. When [3H]Phe-tRNAPhe.EF-Tu- [35S]GTPyS ternary complex (2.6 pmol in 12 pl) was incubated with poly(u)-programmed ribosomes (20 pmol Time in 48 pl), the amount of 3H and 35S bound to filters after 30 s indicated that 0.59 pmol of [3H]Phe-tRNA and only 0.17 pmol of [35S] GTPyS were bound to the ribosomes. Analysis of the nitrocellulose filtrate and the unfiltered reaction mixture for [3sS] thiophosphate indicated the hydrolysis of only and pmol of GTP& respectively. We conclude that GTPyS hydrolysis is not stimulated by nitrocellulose filtration, and that the low level of filter-bound GTPyS is due to the nucleotide or the EF-Tu. nucleotide complex being washed off the ribosome. EF-Tu. aa-trna. GTPyS complex after it binds to the filter. Yokosawa et al. (13) have reported previously that EF-Tu and GMPPNP can be removed from a ribosome. PhetRNA. EF-Tu. GMPPNP complex without hydrolysis during sucrose gradient centrifugation. The Complex between Phe-tRNAph. EF-Tu. GTPyS and Poly(U)-programmed Ribosomes Is an Intermediate in Peptide BondFormation-We have shown previously (7) that the formation of the RS.TC complex, as evidenced by the appearance of nitrocellulose-filterable Phe-tRNA, is as rapid for ternary complexes containing GTPyS as it is for ternary complexes containing GTP. However, there is a disparity in the rate of nucleotide hydrolysis; it is at least 2500-fold slower with GTPyS than with GTP. Evidence that even this slow hydrolysis reaction requires the preliminary formation of the RS.TC complex is provided in Table 11, which shows that GTPyS hydrolysis is dependent on the presence of ribosomes and EF-Tu and is stimulated by the presence of the cognate TABLE I1 Requirements for GTPySase activity Binary complex was prepared by centrifuging a mixture of [35S] GTPyS (210 pmol) and EF-Tu (420 pmol) through a 0.5-ml Bio-Gel P-6 column (see "Materials and Methods"). Ternary complexes were prepared by adding 3-pl aliquots containing 2.8 pmol of [35S]GTPyS to 60 pmol of either Phe-tRNAPhe or Val-tRNAVd in a volume of 18 pl. Where incubation with ribosomes is indicated, 12 pl of AcPhetRNA, poly(u)-programmed ribosomes (22.5 pmol) were added and the samples were analyzed for thiophosphate 8 min later. The complete system is the Phe ternary complex plus ribosomes. For the complete system-ef-tu the Bio-Gel step was omitted. Reactants [35S]Thiophosphate pmol Complete system 1.68 Complete system-ribosomes <0.03 Complete system-ef-tu <0.03 Complete system-phe-trnaphe 0.63 Complete system-phe-trnaphe + Val-tRNAVa' 0.30 Complete system" 2.12 Complete svstem + thiostredton (20 um)" 0.48 "Phe ternary complex (7.4 pmol), prepared as described under "Materials and Methods," and AcPhe-tRNA, poly(u)-programmed ribosomes (40 pmol) were incubated in a volume of 20 pl for 1 min and analyzed for thiophosphate. For the complete system + thiostrepton, 400 pmol of the antibiotic were added to the ribosomes prior to the addition of the ternary complex. x r 2 6 IO (min.) Time (mid FIG. 2. Chase of radioactive GTPyS ternary complex from the ribosome as determined by the peptidyl transfer assay. A, solid circles, AcPhe-tRNA, poly(u)-programmed ribosomes (160 pmol) were mixed with [3H]Phe-tRNA.EF-Tu.[35S]GTPyS complex (30 pmol) in a volume of 80 pl at 5 "C. Samples (4 pl) were quenched in EDTA and analyzed for the formation of peptide. Open circles, as above except that 380 pl Phe-tRNAPhe.EF-Tu.GTPyS complex (380 pmol) was added at 6 s to 20 pl of the labeled ternary complex/ ribosome mixture and 20 p1 samples were quenched in EDTA and analyzed for the formation of peptide. Triangles, as above except that the two complexes were mixed prior to mixing with ribosomes. B, the data from A are plotted as first order plots. The straight lines drawn are regression lines. kz calculated from the ty, of the reaction in the absence of the chase is 4.1 X s-'. k-l + ka calculated from the ti/% of the reaction in the presence of the unlabeled ternary complex chase is 6.3 X s-'. Results plotted reflect the reaction of 1 pl of the labeled ternary complex/ribosome mixture. Phe-tRNA but not by the noncognate Val-tRNA. Hydrolysis is also inhibited by thiostrepton, an antibiotic which binds to the bacterial ribosome and inhibits ternary complex binding to the ribosomal A-site (14). Only the low rate of GTPyS hydrolysis makes it difficult to accept that this reaction is analogous to GTP hydrolysis in the normally rapid reactions of protein biosynthesis. To examine this relationship further we determined the rate of peptide bond formation when the ternary complex contained GTPyS in place of GTP. The reaction showed first order kinetics (Fig. 2) and a rate constant of 4.1 X s-l. This

4 rate is 100-fold slower than the rate of peptidyl transfer (6) in an analogous reaction containing GTP but isimilar to the rate constant of 3.9 X 10-~ s" we have previously reported (7) for the hydrolysis of [35S]GTPyS. The coincidence of the rate constants for GTPyS hydrolysis and peptidyl transfer implies a common rate-determining step. Other results (6, 15, 16) indicate that GTP hydrolysis must precede peptide bond formation. Since peptide bond formation, an event obviously related to protein synthesis, requires prior hydrolysis of GTP-& we believe that the hydrolysis, despite its low rate, is analogous to the hydrolysis which occurs with ternary complexes containing GTP and serves the same function. Use of GTPyS to Study the Accuracy of Protein Biosynthesis The substitution of GTPyS for GTP, by lowering the rate of the GTPase associated with aa-trna binding to ribosomes, has allowed us to determine the rate of noncovalent dissociation of the RS. TC complex, a reaction of great importance to the overall fidelity of protein biosynthesis (7). The principle of the method is to form a 35S-labeled RS. TC complex and determine the rate of GTPyS hydrolysis from that complex (k2) and from an analogous complex in the presence of a chase of unlabeled ternary complex. The function of the chase is to compete with, and thereby inhibit, the formation of labeled RS.TC by 35S-labeled TC which dissociates from the ribosomes. This loss of labeled TC through reversal of the initial binding reaction is reflected in a reduced yield of [35S]thiophosphate and a smaller tth for the reaction. The observed rate constant for GTPyS hydrolysis is equal to k-l + k,; L l, the rate constant for RS. TC dissociation, can be calculated by subtracting from the observed rate constant the value of kz obtained above. In principle, the same measurement could be made if the radiolabel was in the amino acid and the formation of labeled peptide was measured. We have carried out this experiment (Fig. 2) by making a ribosome. GTPyS. EF-Tu. [3H]PhetRNA complex and measuring the rate at which 3H-labeled peptide is formed in the presence and absence of excess EFTu. CTPyS. Phe-tRNA complex. The k-l is 2.2 X s-l, in good agreement with the value obtained from GTP+ hydrolysis (7). The agreement between the k-l determined by chasing the radioactive GTPyS and that determined by chasing the radioactive phe-trna indicates that alternative reactions leading to breakdown of the RS. TC complex, in which the aatrna, EF-Tu. GTPyS, or GTPyS components of the ternary complex leave the ribosome independently, are much slower than that in which the ternary complex leaves as a unit. The same conclusion can be drawn from the observation2 that neither GTPyS nor Phe-tRNA alone can chase their labeled counterparts from the RS. TC complex. To understand how temperature and polycation concentration influence the fidelity of protein biosynthesis (17,18), we have used the methods described earlier to study the effect of these parameters on k-l and kz. Effect of Temperature and Polycations on the Rate of GTPyS Hydrolysis in RS. TC (k&"the rate constant for GTPyS hydrolysis in cognate RS. TC complexes was determined from the progress of [35S]GTPyS hydrolysis when cognate [3H] Phe-tRNA. EF-Tu. [35S]GTP-yS complexes were reacted with excess poly(u)-programmed ribosomes. The effect of temperature on kz in reactions with 5 mm Mgz+ as the only polycation is shown in Table 111. As expected, kz increased as the temperature was raised from 5 to 25 "C. The activation energy D. B. Dix, unpublished results. Accuracy of Ternary Complex Selection by Ribosomes 3241 TABLE IrI Effect of temperature and ionic conditions on k-1 and kz for the reaction between poly(u)-programmed ribosomes and a Phe ternary complex containing GTPyS AcPhe-tRNAPh', poly(u)-programmed ribosomes (200 pmol), were mixed with [3H]Phe-tRNAPh'.EF-Tu.[35S]GTP-yS complex (45 pmol) in a volume of 80 ~1 at the indicated temperature in 50 mm Tris.HCI (ph 7.21, 50 mm KCI, 1 mm dithiothreitol and the M F and spermidine concentrations indicated. Samples (4 pl) were quenched in EDTA and analyzed for [35S]thiophosphate. h2 was determined from the progress of the observed first order reaction. For the determination of k-l, 380 p1 of Phe-tRNAPhe.EF-Tu.GTPyS complex (380 pmol) was added at 6 s to 20 pl of the labeled ternary complex/ribosome mixture. Samples (20 pl) were quenched in EDTA and analyzed for [35S]thiophosphate. The observed first order rate constant for GTP-yS hydrolysis in this reaction is k-] + kz. k-, was calculated by subtracting kz. Temperature Mg2f Spermidine k, X lo3 s-' L 1 X lo3 s-' k&, "C mm mm " <1 > ' 0.16 a In this sample, the rapid progress of the chased reaction did not permit the collection of accurate kinetic data. k l was therefore calculated from the extents of the reactions in the presence and absence of the chase, the ratio of which is kz/(k-, + k2). *Because k-, is much larger than kz, the end point for this reaction is not very much larger than a background reaction where both labeled and unlabeled ternary complex were added together. A drift in the end point that is approximately parallel to that background was subtracted. for the hydrolysis as calculated from an Arrhenius plot (Fig. 3A) is 14 kcal/mol. The effect of polycations on k2 was determined by comparing the rates of reaction in 5 mm Mg" with that in 10 m~ Mg"', and that in 1 mm M e and 1 mm spermidine. The reaction in 10 mm Mg"+ was run at 7 "c and had a first order rate constant of 1.1 X lod2 s-'. This is about a %fold increase over the value of k2 for the reaction in 5 mm Mg2+ and 7 "C (obtained by interpolation between the k2 at 5 and 10 "C). In 1 mm Mg2+ and 1 mm spermidine, h, at 5 "C is 2.7 X s-', which is slightly lower than the comparable rate constant at 5 mm Me. Effect of Temperature and Zonic Conditions on the Rate of RS. TC Dissociation (k-,)-when [3H]Phe-tRNAPhe. EF-Tu. [Y3]GTPyS ternary complex is reacted with a slight excess of poly(u)-programmed ribosomes, and a large excess of unlabeled ternary complex added as a chase seconds after mixing the ribosomes with the labeled complex, a first order reaction is observed. The rate of this reaction is k-i + k2 (7). First order rate constants obtained this way were used to calculate k-, (Table 111) at temperatures and polycation concentrations where k2 was determined in parallel experiments. For the reactions conducted in 5 mm Mg2+, k-, increases with temperature, and this effect of temperature on ktl is substantially greater than that on kz (Table 111). The activation energy for RS. TC dissociation calculated from an Arrhenius plot (Fig. 3B) is 37 kcallmol. The effect of polycations on ky1 is shown in Table 111. Mgz+ at 10 mm concentration appears to cause a large decrease in k-l as evidenced by the failure of the excess unlabeled ternary complex to reduce by any significant amount the labeled ternary complex bound to the ribosomes. In contrast, the dissociation reaction at 1 mm M e and 1 mm spermidine (kl X lo-' s-') is 6-fold faster than at 5 mm M$+.

5 + k Accuracy of Ternary Complex Selection by Ribosomes X t - II) 1-2'6 I r \ t.4 - I \ \ /Tx103 (OK) B I/Ta103 ('K) FIG. 3. Arrhenius plots for nucleotide hydrolysis and ternary complex dissociation from RS*TC complexes containing GTPrS. A, log kp (Table 111) is plotted uersus 1/T. B, log k-i (Table 111) is plotted uersw 1/T. DISCUSSION The similarity of the reactions leading to peptide formation using ternary complexes containing either GTP or GTPyS is strong evidence for similar if not identical reaction pathways. The slower rate of pyrophosphate bond hydrolysis for complexes containing GTPyS probably reflects a lower reactivity of the thiophosphate group participating in the hydrolytic reaction. Slower hydrolysis has been observed in numerous enzymatic systems where thiophosphate replaced the y-phosphate group of nucleotide triphosphates (19, 20). For these reasons we think it likely that factors which change the rate of GTPyS hydrolysis will also affect GTP hydrolysis and that a study of the former reaction can give useful information about the latter, for which direct experimental study is difficult. The similarities of the reaction of GTP and GTPyS also allow us to extend with confidence our findings concerning the rate constant for noncovalent dissociation of the RS. TC complex containing GTPyS, to the analogous reaction of the more interesting complexes containing GTP. The rate constant for this reaction is experimentally inaccessible at this time. Much of the current work on the elongation cycle of protein biosynthesis is concerned with defining the fidelity and speed of this process and the way in which these parameters are related. These discussions are best conducted with a knowledge of the rate constants for the individual reactions that make up the cycle. A good deal of the work of this laboratory has been directed towards measuring the rate constants for the binding of aa-trnas to ribosomes, and the formation of peptide bonds. Despite recent statements to the contrary (21, 22), the available experimental evidence indicates that this latter step is effectively irreversible and independent of the translocation reaction that follows. The mechanism which best accounts for the available data is as follows. RS k Pi -Ik2 aa-trna k3 aa-trna pep-trna + & RS.TC 3 RS -+ RS -+ RS TC k-, EF-Tu:GDP + EF-Tu. GDP k4 Our results show that the interactions of GTPyS with EF- I aa-trna Tu, aa-trna, and ribosomes are very similar to those reported for GTP.in those reactions which lead to the formation of the RS + RS peptide bond. Thus, GTPyS is shown to form a binary com- EF-TU-GDP + plex with EF-Tu and a ternary complex with EF-Tu and Phe- EF-Tu. GDP trna. In addition, the nucleotide is necessary for EF-Tucatalyzed binding of Phe-tRNA to poly(u)-programmed ri- Based on this mechanism, it is clear that the most rapid bosomes. rate of protein synthesis is achieved when none of the correct We have shown previously that GTPyS hydrolysis which ternary complex is rejected, i.e. when kz is much greater than accompanies the reaction between a Phe ternary complex and k-l for the cognate complex. Optimal fidelity, on the other poly(u)-programmed ribosomes is at least 2500-fold slower hand requires that kz be much less than kl for near-cognate than the comparable reaction with GTP (6). Here we have ternary complexes. Experimental work at 5 "c and 5 mm provided further evidence that this slow hydrolysis is neces- M%+ has shown that the value of 122 does not differ greatly sary for binding of Phe-tRNA to ribosomes. Hydrolysis is between cognate and near-cognate complexes (5, 6, 24). The dependent on the presence of ribosomes and EF-Tu, does not reasons for this lack of specificity are only now beginning to occur with a noncognate Val ternary complex, and is inhibited be under~tood.~ However, this finding does imply that maxiby thiostrepton. Furthermore, we show that the rate of peptide mum fidelity and maximum speed of aa-trna selection are formation, which can only occur after pyrophosphate bond mutually incompatible, even though k-1 for the cognate comhydrolysis (5, 15, 16), has been reduced to a rate, 4.1 X plex is four orders of magnitude lower than that for nears-i, that is similar to that for GTPyS hydrolysis. We conclude cognate complexes. The absolute value of & under these that the hydrolysis of GTPyS, despite its low rate, is analo- conditions is so much greater than k-1 for the cognate species gous to the hydrolysis of GTP which normally accompanies that it is comparable to k+ for the near-cognate complex, aa-trna binding to the ribosome, and that it results in the formation of an intermediate in the pathway leading to the R. C. Thompson, D. B. Dix, and A. M. Karim, manuscript in formation of the peptide bond. preparation.

6 I I." resulting in a significant fraction of the near-cognate complexes being accepted by the ribosome. It follows that under these condkions the accuracy of aa-trna selection is sacrificed in favor of speed. The overall accuracy of the process is ensured only by the subsequent proofreading step of aa-trna binding. The suggestion that accuracy and speed might be inversely correlated originated with Ninio (23). The evidence presented in favor of this hypothesis in our earlier paper has lead to a considerable debate (22) but little in the way of data to refute the main points of our argument. The present work was carried out in part to examine whether changing the temperature and polycation concentrations affects the kinetic parameters of initial recognition of ternary complexes by E. coli ribosomes in such a way as to invalidate our previous conclusions that optimal speed and accuracy of protein biosynthesis appear to be mutually exclusive objectives. Dealing first with the effects of temperature, we find that both k-, and k2 (GTPyS) for the cognate ternary complex increase with increasing temperature. The energy of activation for kz when GTPyS is being hydrolyzed is very similar to that observed when GTP is hydrolyzed (24) so we feel the following conclusions apply equally to both reactions. We find that kwl increases faster than kz and that the ratio of kz/k-l is 0.1 for GTPyS and 500 for GTP at 25 "C. Extrapolated to 37 "C the ratios should be 0.02 and 100, respectively. These values should be compared to ratios of 1.4 and 10,000, respectively, at 5 "C. Obviously, the use of low temperatures in our earlier work emphasized the difference between k-, and kz, but equally obviously, the dominant rate constant with GTP as the substrate is k2 even at 37 "C. The question of the appropriate concentration of polycations in an in vitro protein synthesis system is much more difficult to decide since the concentration of free Mg2+ in E. coli has not yet been satisfactorily determined. For this reason we have chosen to determine the effects on the ratio kz/k+ of the lowest concentrations of polycations that will support the binding of aa-trnas to ribosomes. Under these conditions, 1 mm M$+ and 1 mm spermidine, the ratio k2/k-l decreases about 10-fold. At 37 "C and the most extreme polycation concentrations, it appears that the ratio kz/k-, for GTP as substrate will be about 10. The ribosome may therefore be expected to reject less than 1 in 10 of the correct ternary complexes despite the considerable loss of fidelity that this entails. Therefore, the present data support our original hypothesis that maximal speed and maximal accuracy are mutually exclusive in aatrna binding to E. coli ribosomes. Furthermore, the finding that the ratio of k,jk-l under these conditions is about 10 is consistent with the idea that the requirement for speed is the primary factor leading to reduced accuracy. A value of that was much less than one-tenth & would lead to further decreases in accuracy without a significant increase in speed. A ratio of kz to k-, equal to 10 or more is far from typical of enzymatic reactions. k-l is usually larger than & and this is especially likely to be true where the fidelity of substrate selection is of considerable importance to the cell. Despite Accuracy of Ternary Complex Selection by Ribosomes 3243 this, the kinetic constants of the initial recognition step of aa-trna binding to ribosomes seem to facilitate binding of the cognate species rather than the rejection of its nearcognate relatives. It is hard to disassociate this finding from the fact that protein biosynthesis requires a much larger proportion of the cell's mass than any other step of gene expression. The choice of rate constants for maximal speed rather than accuracy may well represent the best choice where so much of the cell's resources are committed. This idea is consistent with the existence of mutants in which the fidelity of protein biosynthesis is higher than that of the wild type, but the organism as a whole is less fit. REFERENCES 1. Uhlenbeck 0. C., Martin, F. H., anddoty, P. (1971) J. Mol. Biol. 57, Thompson, R. C., and Stone, P. M. (1977) Proc. Natl. Acad. Sci. U. S. A. 74, Thompson, R. C., Dix, D. B., Gerson, R. B., and Karim, A. M. (1981) J. Bid. Chem. 256, Hopfield, J. J. (1974) Proc. Natl. Acad. Sei. U. S. A. 71, Thompson, R. C., and Dix, D.B. (1982) J. Biol. Chem. 257, Thompson, R. C., Dix, D. B., and Eccleston, J. F. (1980) J. Bid. Chem. 255, Thomason, R. C., and Karim, A. M. (1982) Proc. 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Bid. 8, Bagshaw, C.R., Eccleston, J. F., Eckstein, F., Goody, R. S., Gutsfreund, H. and Trentham, D. R. (1974) Biochem. J. 141, Eckstein, F. (1975) Angew. Chem. Int. Ed. Engl. 14, Kurland, C.G. (1980) in Ribosomes-Structure, Function, and Genetics (Chambliss, G., Craven, G. R., Davies, J., Davis, K., Kahan, L.. and Nomura. M.. eds) DU Universitv Park Press, Baltimore 22. Kurland. C. G.. and Ehrenburn, M. (1984) in Prowess in Nucleic Acid Research and Molecula? Biology (Cohn, W. E., and Moldave, K., eds) Vol. 31, pp , Academic Press, Inc., Orlando, FL 23. Ninio, J. (1974) J. Mol. Biol. 84, Eccleston, J. F., Dix, D. B., and Thompson, R. C. (1985) J. Biol. Chem. 260,