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. 1997 Jun 15;25(12):2254–2258. doi: 10.1093/nar/25.12.2254

Eukaryotic release factor 1 (eRF1) abolishes readthrough and competes with suppressor tRNAs at all three termination codons in messenger RNA.

G Drugeon 1, O Jean-Jean 1, L Frolova 1, X Le Goff 1, M Philippe 1, L Kisselev 1, A L Haenni 1
PMCID: PMC146740  PMID: 9171074

Abstract

It is known from experiments with bacteria and eukaryotic viruses that readthrough of termination codons located within the open reading frame (ORF) of mRNAs depends on the availability of suppressor tRNA(s) and the efficiency of termination in cells. Consequently, the yield of readthrough products can be used as a measure of the activity of polypeptide chain release factor(s) (RF), key components of the translation termination machinery. Readthrough of the UAG codon located at the end of the ORF encoding the coat protein of beet necrotic yellow vein furovirus is required for virus replication. Constructs harbouring this suppressible UAG codon and derivatives containing a UGA or UAA codon in place of the UAG codon have been used in translation experiments in vitro in the absence or presence of human suppressor tRNAs. Readthrough can be virtually abolished by addition of bacterially-expressed eukaryotic RF1 (eRF1). Thus, eRF1 is functional towards all three termination codons located in a natural mRNA and efficiently competes in vitro with endogenous and exogenous suppressor tRNA(s) at the ribosomal A site. These results are consistent with a crucial role of eRF1 in translation termination and forms the essence of an in vitro assay for RF activity based on the abolishment of readthrough by eRF1.

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Selected References

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  1. Beaudet A. L., Caskey C. T. Mammalian peptide chain termination. II. Codon specificity and GTPase activity of release factor. Proc Natl Acad Sci U S A. 1971 Mar;68(3):619–624. doi: 10.1073/pnas.68.3.619. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Björnsson A., Mottagui-Tabar S., Isaksson L. A. Structure of the C-terminal end of the nascent peptide influences translation termination. EMBO J. 1996 Apr 1;15(7):1696–1704. [PMC free article] [PubMed] [Google Scholar]
  3. Boyer J. C., Drugeon G., Séron K., Morch-Devignes M. D., Agnès F., Haenni A. L. In vitro transcripts of turnip yellow mosaic virus encompassing a long 3' extension or produced from a full-length cDNA clone harbouring a 2 kb-long PCR-amplified segment are infectious. Res Virol. 1993 Sep-Oct;144(5):339–348. doi: 10.1016/s0923-2516(06)80049-x. [DOI] [PubMed] [Google Scholar]
  4. Buckingham R. H. Codon context and protein synthesis: enhancements of the genetic code. Biochimie. 1994;76(5):351–354. doi: 10.1016/0300-9084(94)90108-2. [DOI] [PubMed] [Google Scholar]
  5. Capone J. P., Sharp P. A., RajBhandary U. L. Amber, ochre and opal suppressor tRNA genes derived from a human serine tRNA gene. EMBO J. 1985 Jan;4(1):213–221. doi: 10.1002/j.1460-2075.1985.tb02338.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Caskey C. T., Beaudet A. L., Tate W. P. Mammalian release factor; in vitro assay and purification. Methods Enzymol. 1974;30:293–303. doi: 10.1016/0076-6879(74)30032-8. [DOI] [PubMed] [Google Scholar]
  7. 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]
  8. Chomczynski P., Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem. 1987 Apr;162(1):156–159. doi: 10.1006/abio.1987.9999. [DOI] [PubMed] [Google Scholar]
  9. Frolova L., Le Goff X., Rasmussen H. H., Cheperegin S., Drugeon G., Kress M., Arman I., Haenni A. L., Celis J. E., Philippe M. A highly conserved eukaryotic protein family possessing properties of polypeptide chain release factor. Nature. 1994 Dec 15;372(6507):701–703. doi: 10.1038/372701a0. [DOI] [PubMed] [Google Scholar]
  10. 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]
  11. Goelet P., Lomonossoff G. P., Butler P. J., Akam M. E., Gait M. J., Karn J. Nucleotide sequence of tobacco mosaic virus RNA. Proc Natl Acad Sci U S A. 1982 Oct;79(19):5818–5822. doi: 10.1073/pnas.79.19.5818. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Goldstein J. L., Beaudet A. L., Caskey C. T. Peptide chain termination with mammalian release factor. Proc Natl Acad Sci U S A. 1970 Sep;67(1):99–106. doi: 10.1073/pnas.67.1.99. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. 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]
  14. Haeberlé A. M., Stussi-Garaud C., Schmitt C., Garaud J. C., Richards K. E., Guilley H., Jonard G. Detection by immunogold labelling of P75 readthrough protein near an extremity of beet necrotic yellow vein virus particles. Arch Virol. 1994;134(1-2):195–203. doi: 10.1007/BF01379118. [DOI] [PubMed] [Google Scholar]
  15. 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]
  16. Kawakami K., Nakamura Y. Autogenous suppression of an opal mutation in the gene encoding peptide chain release factor 2. Proc Natl Acad Sci U S A. 1990 Nov;87(21):8432–8436. doi: 10.1073/pnas.87.21.8432. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Kisselev L. L., Frolova LYu Termination of translation in eukaryotes. Biochem Cell Biol. 1995 Nov-Dec;73(11-12):1079–1086. doi: 10.1139/o95-116. [DOI] [PubMed] [Google Scholar]
  18. 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]
  19. McCaughan K. K., Brown C. M., Dalphin M. E., Berry M. J., Tate W. P. Translational termination efficiency in mammals is influenced by the base following the stop codon. Proc Natl Acad Sci U S A. 1995 Jun 6;92(12):5431–5435. doi: 10.1073/pnas.92.12.5431. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. 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]
  21. Morch M. D., Drugeon G., Szafranski P., Haenni A. L. Proteolytic origin of the 150-kilodalton protein encoded by turnip yellow mosaic virus genomic RNA. J Virol. 1989 Dec;63(12):5153–5158. doi: 10.1128/jvi.63.12.5153-5158.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Nakamura Y., Ito K., Isaksson L. A. Emerging understanding of translation termination. Cell. 1996 Oct 18;87(2):147–150. doi: 10.1016/s0092-8674(00)81331-8. [DOI] [PubMed] [Google Scholar]
  23. Niesbach-Klösgen U., Guilley H., Jonard G., Richards K. Immunodetection in vivo of beet necrotic yellow vein virus-encoded proteins. Virology. 1990 Sep;178(1):52–61. doi: 10.1016/0042-6822(90)90378-5. [DOI] [PubMed] [Google Scholar]
  24. 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]
  25. Pelham H. R. Leaky UAG termination codon in tobacco mosaic virus RNA. Nature. 1978 Mar 30;272(5652):469–471. doi: 10.1038/272469a0. [DOI] [PubMed] [Google Scholar]
  26. Quillet L., Guilley H., Jonard G., Richards K. In vitro synthesis of biologically active beet necrotic yellow vein virus RNA. Virology. 1989 Sep;172(1):293–301. doi: 10.1016/0042-6822(89)90131-1. [DOI] [PubMed] [Google Scholar]
  27. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Schmitt C., Balmori E., Jonard G., Richards K. E., Guilley H. In vitro mutagenesis of biologically active transcripts of beet necrotic yellow vein virus RNA 2: evidence that a domain of the 75-kDa readthrough protein is important for efficient virus assembly. Proc Natl Acad Sci U S A. 1992 Jul 1;89(13):5715–5719. doi: 10.1073/pnas.89.13.5715. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Skuzeski J. M., Nichols L. M., Gesteland R. F., Atkins J. F. The signal for a leaky UAG stop codon in several plant viruses includes the two downstream codons. J Mol Biol. 1991 Mar 20;218(2):365–373. doi: 10.1016/0022-2836(91)90718-l. [DOI] [PubMed] [Google Scholar]
  30. 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]
  31. Stansfield I., Jones K. M., Kushnirov V. V., Dagkesamanskaya A. R., Poznyakovski A. I., Paushkin S. V., Nierras C. R., Cox B. S., Ter-Avanesyan M. D., Tuite M. F. The products of the SUP45 (eRF1) and SUP35 genes interact to mediate translation termination in Saccharomyces cerevisiae. EMBO J. 1995 Sep 1;14(17):4365–4373. doi: 10.1002/j.1460-2075.1995.tb00111.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Tamada T., Kusume T. Evidence that the 75K readthrough protein of beet necrotic yellow vein virus RNA-2 is essential for transmission by the fungus Polymyxa betae. J Gen Virol. 1991 Jul;72(Pt 7):1497–1504. doi: 10.1099/0022-1317-72-7-1497. [DOI] [PubMed] [Google Scholar]
  33. Tate W. P., Poole E. S., Mannering S. A. Hidden infidelities of the translational stop signal. Prog Nucleic Acid Res Mol Biol. 1996;52:293–335. doi: 10.1016/s0079-6603(08)60970-8. [DOI] [PubMed] [Google Scholar]
  34. Valle R. P., Drugeon G., Devignes-Morch M. D., Legocki A. B., Haenni A. L. Codon context effect in virus translational readthrough. A study in vitro of the determinants of TMV and Mo-MuLV amber suppression. FEBS Lett. 1992 Jul 20;306(2-3):133–139. doi: 10.1016/0014-5793(92)80984-o. [DOI] [PubMed] [Google Scholar]
  35. Zaccomer B., Haenni A. L., Macaya G. The remarkable variety of plant RNA virus genomes. J Gen Virol. 1995 Feb;76(Pt 2):231–247. doi: 10.1099/0022-1317-76-2-231. [DOI] [PubMed] [Google Scholar]
  36. 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]
  37. ten Dam E. B., Pleij C. W., Bosch L. RNA pseudoknots: translational frameshifting and readthrough on viral RNAs. Virus Genes. 1990 Jul;4(2):121–136. doi: 10.1007/BF00678404. [DOI] [PMC free article] [PubMed] [Google Scholar]

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