Skip to main content
PMC home page
Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1982 Aug;79(16):4922–4926. doi: 10.1073/pnas.79.16.4922

The accuracy of protein biosynthesis is limited by its speed: high fidelity selection by ribosomes of aminoacyl-tRNA ternary complexes containing GTP[gamma S]

R C Thompson, A M Karim
PMCID: PMC346797  PMID: 6750613

Abstract

Guanosine 5'-[gamma-thio]triphosphate (GTP[gamma S] ) forms a stable ternary complex with polypeptide chain elongation factor Tu (EF-Tu) and aminoacyl-tRNA, and this complex binds rapidly and tightly to a properly programmed ribosome. However, the rate constant for the subsequent hydrolysis of the beta-gamma pyrophosphate bond (3.9 X 10(-3) s-1 at 5 degrees C) is less than 1/2,500th of that for the analogous reaction of GTP. We have taken advantage of this low rate to determine the rate constant for dissociation of the complex of poly(U)-programed ribosomes, EF-Tu, Phe-tRNAPhe, and GTP[gamma S] (2.7 X 10(-3) s-1) and the second-order rate constant for formation of this complex (3.3 X 10(6) M-1 s-1). Therefore, the Kd of the complex may be calculated to be 8.2 X 10(-10) M. An analogous near-cognate complex with Leu-tRNA2Leu in place of Phe-tRNAPhe has been determined by equilibrium methods to have a Kd greater than 1.7 X 10(-6) M. These results indicate that under equilibrium conditions the ribosome can distinguish cognate and near-cognate ternary complexes with great accuracy. Therefore, its failure to show this high specificity with the physiological ternary complexes containing GTP is due to the speed of GTP hydrolysis being similar to the speed of dissociation of the near-cognate complex. The low specificity of the physiological reaction is corrected by subsequent proofreading. The results reported here suggest that proofreading is necessary not simply for high accuracy but for the combination of speed and accuracy required in protein biosynthesis.

Full text

PDF
4922

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Bagshaw C. R., Eccleston J. F., Eckstein F., Goody R. S., Gutfreund H., Trentham D. R. The magnesium ion-dependent adenosine triphosphatase of myosin. Two-step processes of adenosine triphosphate association and adenosine diphosphate dissociation. Biochem J. 1974 Aug;141(2):351–364. doi: 10.1042/bj1410351. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Blomberg C. A kinetic recognition process for tRNA at the ribosome. J Theor Biol. 1977 May 21;66(2):307–325. doi: 10.1016/0022-5193(77)90174-6. [DOI] [PubMed] [Google Scholar]
  3. Cassidy P. S., Kerrick W. G. Enzymatic preparation of adenosine 5'-O-(3-[35S]thiotriphosphate) by thiophosphorylation of adenosine diphosphate. Biochim Biophys Acta. 1979 Nov 22;565(1):209–213. doi: 10.1016/0005-2787(79)90097-2. [DOI] [PubMed] [Google Scholar]
  4. Eccleston J. F., Messerschmidt R. G., Yates D. W. A simple rapid mixing device. Anal Biochem. 1980 Jul 15;106(1):73–77. doi: 10.1016/0003-2697(80)90120-7. [DOI] [PubMed] [Google Scholar]
  5. Eckstein F. Investigation of enzyme mechanisms with nucleoside phosphorothioates. Angew Chem Int Ed Engl. 1975 Mar;14(3):160–166. doi: 10.1002/anie.197501601. [DOI] [PubMed] [Google Scholar]
  6. Edelmann P., Gallant J. Mistranslation in E. coli. Cell. 1977 Jan;10(1):131–137. doi: 10.1016/0092-8674(77)90147-7. [DOI] [PubMed] [Google Scholar]
  7. Galas D. J., Branscomb E. W. Ribosome slowed by mutation to streptomycin resistance. Nature. 1976 Aug 12;262(5569):617–619. doi: 10.1038/262617b0. [DOI] [PubMed] [Google Scholar]
  8. Grosjean H. J., de Henau S., Crothers D. M. On the physical basis for ambiguity in genetic coding interactions. Proc Natl Acad Sci U S A. 1978 Feb;75(2):610–614. doi: 10.1073/pnas.75.2.610. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Hopfield J. J. Kinetic proofreading: a new mechanism for reducing errors in biosynthetic processes requiring high specificity. Proc Natl Acad Sci U S A. 1974 Oct;71(10):4135–4139. doi: 10.1073/pnas.71.10.4135. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Kurland C. G. The role of guanine nucleotides in protein biosynthesis. Biophys J. 1978 Jun;22(3):373–392. doi: 10.1016/S0006-3495(78)85494-0. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Laughrea M. Speed-accuracy relationships during in vitro and in vivo protein biosynthesis. Biochimie. 1981 Mar;63(3):145–168. doi: 10.1016/s0300-9084(81)80189-7. [DOI] [PubMed] [Google Scholar]
  12. Loftfield R. B., Vanderjagt D. The frequency of errors in protein biosynthesis. Biochem J. 1972 Aug;128(5):1353–1356. doi: 10.1042/bj1281353. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Lucas-Lenard J., Tao P., Haenni A. L. Further studies on bacterial polypeptide elongation. Cold Spring Harb Symp Quant Biol. 1969;34:455–462. doi: 10.1101/sqb.1969.034.01.051. [DOI] [PubMed] [Google Scholar]
  14. Murthy M. R., Bharucha A. D. Ultracentrifugation of small volumes of tissue extracts without the use of tube adapters. Anal Biochem. 1978 Mar;85(1):251–254. doi: 10.1016/0003-2697(78)90296-8. [DOI] [PubMed] [Google Scholar]
  15. NIRENBERG M., LEDER P. RNA CODEWORDS AND PROTEIN SYNTHESIS. THE EFFECT OF TRINUCLEOTIDES UPON THE BINDING OF SRNA TO RIBOSOMES. Science. 1964 Sep 25;145(3639):1399–1407. doi: 10.1126/science.145.3639.1399. [DOI] [PubMed] [Google Scholar]
  16. Ninio J. A semi-quantitative treatment of missense and nonsense suppression in the strA and ram ribosomal mutants of Escherichia coli. Evaluation of some molecular parameters of translation in vivo. J Mol Biol. 1974 Apr 5;84(2):297–313. doi: 10.1016/0022-2836(74)90586-5. [DOI] [PubMed] [Google Scholar]
  17. Ninio J. Kinetic amplification of enzyme discrimination. Biochimie. 1975;57(5):587–595. doi: 10.1016/s0300-9084(75)80139-8. [DOI] [PubMed] [Google Scholar]
  18. Piepersberg W., Noseda V., Böck A. Bacterial ribosomes with two ambiguity mutations: effects of translational fidelity, on the response to aminoglycosides and on the rate of protein synthesis. Mol Gen Genet. 1979 Mar 9;171(1):23–34. doi: 10.1007/BF00274011. [DOI] [PubMed] [Google Scholar]
  19. Pingoud A., Urbanke C., Krauss G., Peters F., Maass G. Ternary complex formation between elongation factor Tu, GTP and aminoacyl-tRNA: an equilibrium study. Eur J Biochem. 1977 Sep;78(2):403–409. doi: 10.1111/j.1432-1033.1977.tb11752.x. [DOI] [PubMed] [Google Scholar]
  20. Thomposon R. C., Dix D. B. Accuracy of protein biosynthesis. A kinetic study of the reaction of poly(U)-programmed ribosomes with a leucyl-tRNA2-elongation factor Tu-GTP complex. J Biol Chem. 1982 Jun 25;257(12):6677–6682. [PubMed] [Google Scholar]
  21. Thompson R. C., Dix D. B., Eccleston J. F. Single turnover kinetic studies of guanosine triphosphate hydrolysis and peptide formation in the elongation factor Tu-dependent binding of aminoacyl-tRNA to Escherichia coli ribosomes. J Biol Chem. 1980 Dec 10;255(23):11088–11090. [PubMed] [Google Scholar]
  22. Thompson R. C., Dix D. B., Gerson R. B., Karim A. M. A GTPase reaction accompanying the rejection of Leu-tRNA2 by UUU-programmed ribosomes. Proofreading of the codon-anticodon interaction by ribosomes. J Biol Chem. 1981 Jan 10;256(1):81–86. [PubMed] [Google Scholar]
  23. Thompson R. C., Stone P. J. Proofreading of the codon-anticodon interaction on ribosomes. Proc Natl Acad Sci U S A. 1977 Jan;74(1):198–202. doi: 10.1073/pnas.74.1.198. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Uhlenbeck O. C., Martin F. H., Doty P. Self-complementary oligoribonucleotides: effects of helix defects and guanylic acid-cytidylic acid base pairs. J Mol Biol. 1971 Apr 28;57(2):217–229. doi: 10.1016/0022-2836(71)90342-1. [DOI] [PubMed] [Google Scholar]
  25. Yates J. L. Role of ribosomal protein S12 in discrimination of aminoacyl-tRNA. J Biol Chem. 1979 Nov 25;254(22):11550–11554. [PubMed] [Google Scholar]
  26. Yokosawa H., Kawakita M., Arai K., Inoue-Yokosawa N., Kaziro Y. Binding of aminoacyl-tRNA to ribosomes promoted by elongation factor Tu. Studies on the role of GTP hydrolysis. J Biochem. 1975 Apr;77(4):719–728. doi: 10.1093/oxfordjournals.jbchem.a130775. [DOI] [PubMed] [Google Scholar]
  27. Yount R. G. ATP analogs. Adv Enzymol Relat Areas Mol Biol. 1975;43:1–56. doi: 10.1002/9780470122884.ch1. [DOI] [PubMed] [Google Scholar]
  28. Zengel J. M., Young R., Dennis P. P., Nomura M. Role of ribosomal protein S12 in peptide chain elongation: analysis of pleiotropic, streptomycin-resistant mutants of Escherichia coli. J Bacteriol. 1977 Mar;129(3):1320–1329. doi: 10.1128/jb.129.3.1320-1329.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Proceedings of the National Academy of Sciences of the United States of America are provided here courtesy of National Academy of Sciences

RESOURCES