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. 1997 Jul 1;16(13):4126–4133. doi: 10.1093/emboj/16.13.4126

Release factor RF3 in E.coli accelerates the dissociation of release factors RF1 and RF2 from the ribosome in a GTP-dependent manner.

D V Freistroffer 1, M Y Pavlov 1, J MacDougall 1, R H Buckingham 1, M Ehrenberg 1
PMCID: PMC1170035  PMID: 9233821

Abstract

Ribosomes complexed with synthetic mRNA and peptidyl-tRNA, ready for peptide release, were purified by gel filtration and used to study the function of release factor RF3 and guanine nucleotides in the termination of protein synthesis. The peptide-releasing activity of RF1 and RF2 in limiting concentrations was stimulated by the addition of RF3 and GTP, stimulated, though to a lesser extent, by RF3 and a non-hydrolysable GTP analogue, and inhibited by RF3 and GDP or RF3 without guanine nucleotide. With short incubation times allowing only a single catalytic cycle of RF1 or RF2, peptide release activity was independent of RF3 and guanine nucleotide. RF3 hydrolysis of GTP to GDP + P(i) was dependent only on ribosomes and not on RF1 or RF2. RF3 affected neither the rate of association of RF1 and RF2 with the ribosome nor the catalytic rate of peptide release. A model is proposed which explains how RF3 recycles RF1 and RF2 by displacing the factors from the ribosome after the release of peptide.

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Selected References

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

  1. AEvarsson A., Brazhnikov E., Garber M., Zheltonosova J., Chirgadze Y., al-Karadaghi S., Svensson L. A., Liljas A. Three-dimensional structure of the ribosomal translocase: elongation factor G from Thermus thermophilus. EMBO J. 1994 Aug 15;13(16):3669–3677. doi: 10.1002/j.1460-2075.1994.tb06676.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Block S. M. Fifty ways to love your lever: myosin motors. Cell. 1996 Oct 18;87(2):151–157. doi: 10.1016/s0092-8674(00)81332-x. [DOI] [PubMed] [Google Scholar]
  3. Calogero R. A., Pon C. L., Canonaco M. A., Gualerzi C. O. Selection of the mRNA translation initiation region by Escherichia coli ribosomes. Proc Natl Acad Sci U S A. 1988 Sep;85(17):6427–6431. doi: 10.1073/pnas.85.17.6427. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Caskey C. T., Tompkins R., Scolnick E., Caryk T., Nirenberg M. Sequential translation of trinucleotide codons for the initiation and termination of protein synthesis. Science. 1968 Oct 4;162(3849):135–138. doi: 10.1126/science.162.3849.135. [DOI] [PubMed] [Google Scholar]
  5. Czworkowski J., Wang J., Steitz T. A., Moore P. B. The crystal structure of elongation factor G complexed with GDP, at 2.7 A resolution. EMBO J. 1994 Aug 15;13(16):3661–3668. doi: 10.1002/j.1460-2075.1994.tb06675.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Frolova L., Le Goff X., Zhouravleva G., Davydova E., Philippe M., Kisselev L. Eukaryotic polypeptide chain release factor eRF3 is an eRF1- and ribosome-dependent guanosine triphosphatase. RNA. 1996 Apr;2(4):334–341. [PMC free article] [PubMed] [Google Scholar]
  7. Ganoza M. C. Polypeptide chain termination in cell-free extracts of E. coli. Cold Spring Harb Symp Quant Biol. 1966;31:273–278. doi: 10.1101/sqb.1966.031.01.035. [DOI] [PubMed] [Google Scholar]
  8. Grentzmann G., Brechemier-Baey D., Heurgue V., Mora L., Buckingham R. H. Localization and characterization of the gene encoding release factor RF3 in Escherichia coli. Proc Natl Acad Sci U S A. 1994 Jun 21;91(13):5848–5852. doi: 10.1073/pnas.91.13.5848. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Grentzmann G., Brechemier-Baey D., Heurgué-Hamard V., Buckingham R. H. Function of polypeptide chain release factor RF-3 in Escherichia coli. RF-3 action in termination is predominantly at UGA-containing stop signals. J Biol Chem. 1995 May 5;270(18):10595–10600. doi: 10.1074/jbc.270.18.10595. [DOI] [PubMed] [Google Scholar]
  10. Holmes K. C. Muscle proteins--their actions and interactions. Curr Opin Struct Biol. 1996 Dec;6(6):781–789. doi: 10.1016/s0959-440x(96)80008-x. [DOI] [PubMed] [Google Scholar]
  11. Ito K., Ebihara K., Uno M., Nakamura Y. Conserved motifs in prokaryotic and eukaryotic polypeptide release factors: tRNA-protein mimicry hypothesis. Proc Natl Acad Sci U S A. 1996 May 28;93(11):5443–5448. doi: 10.1073/pnas.93.11.5443. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Jelenc P. C., Kurland C. G. Nucleoside triphosphate regeneration decreases the frequency of translation errors. Proc Natl Acad Sci U S A. 1979 Jul;76(7):3174–3178. doi: 10.1073/pnas.76.7.3174. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Jelenc P. C. Rapid purification of highly active ribosomes from Escherichia coli. Anal Biochem. 1980 Jul 1;105(2):369–374. doi: 10.1016/0003-2697(80)90472-8. [DOI] [PubMed] [Google Scholar]
  14. Jørgensen F., Adamski F. M., Tate W. P., Kurland C. G. Release factor-dependent false stops are infrequent in Escherichia coli. J Mol Biol. 1993 Mar 5;230(1):41–50. doi: 10.1006/jmbi.1993.1124. [DOI] [PubMed] [Google Scholar]
  15. Kaziro Y. The role of guanosine 5'-triphosphate in polypeptide chain elongation. Biochim Biophys Acta. 1978 Sep 21;505(1):95–127. doi: 10.1016/0304-4173(78)90009-5. [DOI] [PubMed] [Google Scholar]
  16. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  17. Matsumura K., Ito K., Kawazu Y., Mikuni O., Nakamura Y. Suppression of temperature-sensitive defects of polypeptide release factors RF-1 and RF-2 by mutations or by an excess of RF-3 in Escherichia coli. J Mol Biol. 1996 May 17;258(4):588–599. doi: 10.1006/jmbi.1996.0271. [DOI] [PubMed] [Google Scholar]
  18. Mikuni O., Ito K., Moffat J., Matsumura K., McCaughan K., Nobukuni T., Tate W., Nakamura Y. Identification of the prfC gene, which encodes peptide-chain-release factor 3 of Escherichia coli. Proc Natl Acad Sci U S A. 1994 Jun 21;91(13):5798–5802. doi: 10.1073/pnas.91.13.5798. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Mullis K., Faloona F., Scharf S., Saiki R., Horn G., Erlich H. Specific enzymatic amplification of DNA in vitro: the polymerase chain reaction. Cold Spring Harb Symp Quant Biol. 1986;51(Pt 1):263–273. doi: 10.1101/sqb.1986.051.01.032. [DOI] [PubMed] [Google Scholar]
  20. Nissen P., Kjeldgaard M., Thirup S., Polekhina G., Reshetnikova L., Clark B. F., Nyborg J. Crystal structure of the ternary complex of Phe-tRNAPhe, EF-Tu, and a GTP analog. Science. 1995 Dec 1;270(5241):1464–1472. doi: 10.1126/science.270.5241.1464. [DOI] [PubMed] [Google Scholar]
  21. Pavlov M. Y., Ehrenberg M. Rate of translation of natural mRNAs in an optimized in vitro system. Arch Biochem Biophys. 1996 Apr 1;328(1):9–16. doi: 10.1006/abbi.1996.0136. [DOI] [PubMed] [Google Scholar]
  22. Rodnina M. V., Savelsbergh A., Katunin V. I., Wintermeyer W. Hydrolysis of GTP by elongation factor G drives tRNA movement on the ribosome. Nature. 1997 Jan 2;385(6611):37–41. doi: 10.1038/385037a0. [DOI] [PubMed] [Google Scholar]
  23. Seno T., Kobayashi M., Nishimura S. Purification of Excherichia coli methionine tRNAF and methionine tRNAM and studies on their biophysical and biochemical properties. Biochim Biophys Acta. 1968 Nov 20;169(1):80–94. doi: 10.1016/0005-2787(68)90010-5. [DOI] [PubMed] [Google Scholar]
  24. Soffientini A., Lorenzetti R., Gastaldo L., Parlett J. H., Spurio R., La Teana A., Islam K. Purification procedure for bacterial translational initiation factors IF2 and IF3. Protein Expr Purif. 1994 Apr;5(2):118–124. doi: 10.1006/prep.1994.1018. [DOI] [PubMed] [Google Scholar]
  25. Stansfield I., Eurwilaichitr L., Akhmaloka, Tuite M. F. Depletion in the levels of the release factor eRF1 causes a reduction in the efficiency of translation termination in yeast. Mol Microbiol. 1996 Jun;20(6):1135–1143. doi: 10.1111/j.1365-2958.1996.tb02634.x. [DOI] [PubMed] [Google Scholar]
  26. Zhouravleva G., Frolova L., Le Goff X., Le Guellec R., Inge-Vechtomov S., Kisselev L., Philippe M. Termination of translation in eukaryotes is governed by two interacting polypeptide chain release factors, eRF1 and eRF3. EMBO J. 1995 Aug 15;14(16):4065–4072. doi: 10.1002/j.1460-2075.1995.tb00078.x. [DOI] [PMC free article] [PubMed] [Google Scholar]

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