Skip to main content
NCBI 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
. 1976 Apr;73(4):1164–1168. doi: 10.1073/pnas.73.4.1164

Direct experimental evidence for kinetic proofreading in amino acylation of tRNAIle.

J J Hopfield, T Yamane, V Yue, S M Coutts
PMCID: PMC430221  PMID: 1063397

Abstract

Kinetic proofreading is a reaction scheme with a structure more complicated than that of Michaelis kinetics, which leads to a proofreading for errors in the recognition of a correct substrate by an enzyme. We have measured the stoichiometry between ATP hydrolysis and tRNAIle charging, using the enzyme isoleucyl-tRNA synthetase [L-isoleucine:tRNAIle ligase (AMP-forming), EC 6.1.1.5] and the amino acids isoleucine (correct) and valine (incorrect). The enzymatic deacylation of charged tRNA, which would normally prevent meaningful stoichiometry studies, was eliminated by the use of transfer factor Tu-GTP, (which binds strongly to charged tRNA) in the reaction mixture. For isoleucine, 1.5 ATP molecules are hydrolyzed per tRNA charged, but for valine, 270. These stoichiometry ratios are fundamental to kinetic proofreading, for the energy coupling is essential and proofreading is obtained only by departing from 1:1 stoichiometry between energy coupling and product formation. Within the known reaction pathway, these ratios demonstrate that kinetic proofreading induces a reduction in errors by a factor of 1/180. An overall error rate of about 10(-4) for tRNA charging is obtained by a kinetic proofreading using a fundamental discrimination level of about 10(-2), and is compatible with the low in vivo error rate of protein synthesis.

Full text

PDF
1164

Selected References

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

  1. Baldwin A. N., Berg P. Purification and properties of isoleucyl ribonucleic acid synthetase from Escherichia coli. J Biol Chem. 1966 Feb 25;241(4):831–838. [PubMed] [Google Scholar]
  2. Bonnet J. Studies on the mechanism of deacylation of aminoacyl-tRNAs by aminoacyl-tRNA synthetases in the absence of adenosine monophosphate and pyrophosphate. Biochimie. 1974;56(4):541–545. doi: 10.1016/s0300-9084(74)80071-4. [DOI] [PubMed] [Google Scholar]
  3. Calendar R., Berg P. D-Tyrosyl RNA: formation, hydrolysis and utilization for protein synthesis. J Mol Biol. 1967 May 28;26(1):39–54. doi: 10.1016/0022-2836(67)90259-8. [DOI] [PubMed] [Google Scholar]
  4. Eldred E. W., Schimmel P. R. Investigation of the transfer of amino acid from a transfer ribonucleic acid synthetase-aminoacyl adenylate complex to transfer ribonucleic acid. Biochemistry. 1972 Jan 4;11(1):17–23. doi: 10.1021/bi00751a004. [DOI] [PubMed] [Google Scholar]
  5. Eldred E. W., Schimmel P. R. Rapid deacylation by isoleucyl transfer ribonucleic acid synthetase of isoleucine-specific transfer ribonucleic acid aminoacylated with valine. J Biol Chem. 1972 May 10;247(9):2961–2964. [PubMed] [Google Scholar]
  6. Furano A. V. Content of elongation factor Tu in Escherichia coli. Proc Natl Acad Sci U S A. 1975 Dec;72(12):4780–4784. doi: 10.1073/pnas.72.12.4780. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. 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]
  8. Loftfield R. B., Eigner E. A. The specificity of enzymic reactions. Aminoacyl-soluble RNA ligases. Biochim Biophys Acta. 1966 Dec 28;130(2):426–448. doi: 10.1016/0304-4165(66)90239-x. [DOI] [PubMed] [Google Scholar]
  9. 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]
  10. Miller D. L., Weissbach H. Studies on the purification and properties of factor Tu from E. coli. Arch Biochem Biophys. 1970 Nov;141(1):26–37. doi: 10.1016/0003-9861(70)90102-5. [DOI] [PubMed] [Google Scholar]
  11. Modolell J., Vazquez D. Inhibition by aminoacyl transfer ribonucleic acid of elongation factor G-dependent binding of guanosine nucleotide to ribosomes. J Biol Chem. 1973 Jan 25;248(2):488–493. [PubMed] [Google Scholar]
  12. 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]
  13. Pearson R. L., Weiss J. F., Kelmers A. D. Improved separation of transfer RNA's on polychlorotrifuoroethylene-supported reversed-phase chromatography columns. Biochim Biophys Acta. 1971 Feb 11;228(3):770–774. doi: 10.1016/0005-2787(71)90748-9. [DOI] [PubMed] [Google Scholar]
  14. Schreier A. A., Schimmel P. R. Transfer ribonucleic acid synthetase catalyzed deacylation of aminoacyl transfer ribonucleic acid in the absence of adenosine monophosphate and pyrophosphate. Biochemistry. 1972 Apr 25;11(9):1582–1589. doi: 10.1021/bi00759a006. [DOI] [PubMed] [Google Scholar]
  15. Schuber F., Pinck M. On the chemical reactivity of aminoacyl-tRNA ester bond. I. Influence of pH and nature of the acyl group on the rate of hydrolysis. Biochimie. 1974;56(3):383–390. doi: 10.1016/s0300-9084(74)80146-x. [DOI] [PubMed] [Google Scholar]
  16. Skoultchi A., Ono Y., Moon H. M., Lengyel P. On three complementary amino acid polymerization factors from Bacillus stearothermophilus: separation of a complex containing two of the factors, guanosine-5'-triphosphate and aminoacyl-transfer RNA. Proc Natl Acad Sci U S A. 1968 Jun;60(2):675–682. doi: 10.1073/pnas.60.2.675. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Waters L. C., Yang W. K., Mullin B. C., Nichols J. L. Purification of tryptophan transfer RNA from chick cells and its identity with "spot 1" RNA of Rous sarcoma virus. J Biol Chem. 1975 Aug 25;250(16):6627–6629. [PubMed] [Google Scholar]
  18. Yarus M., Berg P. Recognition of tRNA by isoleucyl-tRNA synthetase. Effect of substrates on the dynamics of tRNA-enzyme interaction. J Mol Biol. 1969 Jun 14;42(2):171–189. doi: 10.1016/0022-2836(69)90037-0. [DOI] [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