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. 1998 Jun 1;26(11):2789–2796. doi: 10.1093/nar/26.11.2789

Quantitative analysis of in vivo ribosomal events at UGA and UAG stop codons.

S Mottagui-Tabar 1
PMCID: PMC147583  PMID: 9592169

Abstract

An in vivo translation assay system has been designed to measure, in one and the same assay, the three alternatives for a ribosome poised at a stop codon (termination, read-through and frameshift). A quantitative analysis of the competition has been done in the presence and absence of release factor (RF) mutants, nonsense suppressors and an upstream Shine-Dalgarno-like sequence. The ribosomal +1 frameshift product is measurable when the stop codon is decoded by wild-type or mutant RF (prf A1 or prf B2) and also in the presence of competing suppressor tRNAs. Frameshift frequency appears to be influenced by RF activity. The amount of frameshift product decreases in the presence of competing suppressor tRNAs, however, this decrease is not in proportion to the corresponding increase in the suppression product. Instead, there is an increase in the total amount of protein expressed from the gene, perhaps due to the purging of queued ribosomes. Mutated RFs reduce the total output of the reporter gene by reducing the amount of all three protein products. The nascent peptide has earlier been shown to influence the translation termination process by interacting with the RFs. At 42 degrees C in a temperature-sensitive RF mutant strain, protein measurements indicate that the nascent peptide seems to influence the binding efficiencies of the RFs.

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Selected References

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  1. Adamski F. M., Donly B. C., Tate W. P. Competition between frameshifting, termination and suppression at the frameshift site in the Escherichia coli release factor-2 mRNA. Nucleic Acids Res. 1993 Nov 11;21(22):5074–5078. doi: 10.1093/nar/21.22.5074. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bain J. D., Switzer C., Chamberlin A. R., Benner S. A. Ribosome-mediated incorporation of a non-standard amino acid into a peptide through expansion of the genetic code. Nature. 1992 Apr 9;356(6369):537–539. doi: 10.1038/356537a0. [DOI] [PubMed] [Google Scholar]
  3. Björnsson A., Isaksson L. A. Accumulation of a mRNA decay intermediate by ribosomal pausing at a stop codon. Nucleic Acids Res. 1996 May 1;24(9):1753–1757. doi: 10.1093/nar/24.9.1753. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Björnsson A., Isaksson L. A. UGA codon context which spans three codons. Reversal by ms2i6A37 in tRNA, mutation in rpsD(S4) or streptomycin. J Mol Biol. 1993 Aug 20;232(4):1017–1029. doi: 10.1006/jmbi.1993.1457. [DOI] [PubMed] [Google Scholar]
  5. 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]
  6. Buckingham R. H. Codon context. Experientia. 1990 Dec 1;46(11-12):1126–1133. doi: 10.1007/BF01936922. [DOI] [PubMed] [Google Scholar]
  7. Craigen W. J., Caskey C. T. Expression of peptide chain release factor 2 requires high-efficiency frameshift. Nature. 1986 Jul 17;322(6076):273–275. doi: 10.1038/322273a0. [DOI] [PubMed] [Google Scholar]
  8. Craigen W. J., Cook R. G., Tate W. P., Caskey C. T. Bacterial peptide chain release factors: conserved primary structure and possible frameshift regulation of release factor 2. Proc Natl Acad Sci U S A. 1985 Jun;82(11):3616–3620. doi: 10.1073/pnas.82.11.3616. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Curran J. F. Analysis of effects of tRNA:message stability on frameshift frequency at the Escherichia coli RF2 programmed frameshift site. Nucleic Acids Res. 1993 Apr 25;21(8):1837–1843. doi: 10.1093/nar/21.8.1837. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Curran J. F., Yarus M. Rates of aminoacyl-tRNA selection at 29 sense codons in vivo. J Mol Biol. 1989 Sep 5;209(1):65–77. doi: 10.1016/0022-2836(89)90170-8. [DOI] [PubMed] [Google Scholar]
  11. Curran J. F., Yarus M. Use of tRNA suppressors to probe regulation of Escherichia coli release factor 2. J Mol Biol. 1988 Sep 5;203(1):75–83. doi: 10.1016/0022-2836(88)90092-7. [DOI] [PubMed] [Google Scholar]
  12. Eggertsson G., Söll D. Transfer ribonucleic acid-mediated suppression of termination codons in Escherichia coli. Microbiol Rev. 1988 Sep;52(3):354–374. doi: 10.1128/mr.52.3.354-374.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Faxén M., Kirsebom L. A., Isaksson L. A. Is efficiency of suppressor tRNAs controlled at the level of ribosomal proofreading in vivo? J Bacteriol. 1988 Aug;170(8):3756–3760. doi: 10.1128/jb.170.8.3756-3760.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Goodman H. M., Abelson J., Landy A., Brenner S., Smith J. D. Amber suppression: a nucleotide change in the anticodon of a tyrosine transfer RNA. Nature. 1968 Mar 16;217(5133):1019–1024. doi: 10.1038/2171019a0. [DOI] [PubMed] [Google Scholar]
  15. 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]
  16. Komine Y., Adachi T., Inokuchi H., Ozeki H. Genomic organization and physical mapping of the transfer RNA genes in Escherichia coli K12. J Mol Biol. 1990 Apr 20;212(4):579–598. doi: 10.1016/0022-2836(90)90224-A. [DOI] [PubMed] [Google Scholar]
  17. Komine Y., Kitabatake M., Inokuchi H. 10Sa RNA is associated with 70S ribosome particles in Escherichia coli. J Biochem. 1996 Mar;119(3):463–467. doi: 10.1093/oxfordjournals.jbchem.a021264. [DOI] [PubMed] [Google Scholar]
  18. Kopelowitz J., Hampe C., Goldman R., Reches M., Engelberg-Kulka H. Influence of codon context on UGA suppression and readthrough. J Mol Biol. 1992 May 20;225(2):261–269. doi: 10.1016/0022-2836(92)90920-f. [DOI] [PubMed] [Google Scholar]
  19. 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]
  20. Mikuni O., Kawakami K., Nakamura Y. Sequence and functional analysis of mutations in the gene encoding peptide-chain-release factor 2 of Escherichia coli. Biochimie. 1991 Dec;73(12):1509–1516. doi: 10.1016/0300-9084(91)90185-4. [DOI] [PubMed] [Google Scholar]
  21. Mottagui-Tabar S., Björnsson A., Isaksson L. A. The second to last amino acid in the nascent peptide as a codon context determinant. EMBO J. 1994 Jan 1;13(1):249–257. doi: 10.1002/j.1460-2075.1994.tb06255.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Mottagui-Tabar S., Isaksson L. A. Influence of the last amino acid in the nascent peptide on EF-Tu during decoding. Biochimie. 1996;78(11-12):953–958. doi: 10.1016/s0300-9084(97)86717-x. [DOI] [PubMed] [Google Scholar]
  23. 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]
  24. Parker J., Precup J. Mistranslation during phenylalanine starvation. Mol Gen Genet. 1986 Jul;204(1):70–74. doi: 10.1007/BF00330189. [DOI] [PubMed] [Google Scholar]
  25. Pedersen S. Escherichia coli ribosomes translate in vivo with variable rate. EMBO J. 1984 Dec 1;3(12):2895–2898. doi: 10.1002/j.1460-2075.1984.tb02227.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Pedersen W. T., Curran J. F. Effects of the nucleotide 3' to an amber codon on ribosomal selection rates of suppressor tRNA and release factor-1. J Mol Biol. 1991 May 20;219(2):231–241. doi: 10.1016/0022-2836(91)90564-m. [DOI] [PubMed] [Google Scholar]
  27. Poole E. S., Brown C. M., Tate W. P. The identity of the base following the stop codon determines the efficiency of in vivo translational termination in Escherichia coli. EMBO J. 1995 Jan 3;14(1):151–158. doi: 10.1002/j.1460-2075.1995.tb06985.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Roesser J. R., Yanofsky C. Ribosome release modulates basal level expression of the trp operon of Escherichia coli. J Biol Chem. 1988 Oct 5;263(28):14251–14255. [PubMed] [Google Scholar]
  29. Rydén S. M., Isaksson L. A. A temperature-sensitive mutant of Escherichia coli that shows enhanced misreading of UAG/A and increased efficiency for some tRNA nonsense suppressors. Mol Gen Genet. 1984;193(1):38–45. doi: 10.1007/BF00327411. [DOI] [PubMed] [Google Scholar]
  30. Sandler S. J., Clark A. J. Factors affecting expression of the recF gene of Escherichia coli K-12. Gene. 1990 Jan 31;86(1):35–43. doi: 10.1016/0378-1119(90)90111-4. [DOI] [PubMed] [Google Scholar]
  31. Sipley J., Goldman E. Increased ribosomal accuracy increases a programmed translational frameshift in Escherichia coli. Proc Natl Acad Sci U S A. 1993 Mar 15;90(6):2315–2319. doi: 10.1073/pnas.90.6.2315. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Steege D. A. A nucleotide change in the anticodon of an Escherichia coli serine transfer RNA results in supD-amber suppression. Nucleic Acids Res. 1983 Jun 11;11(11):3823–3832. doi: 10.1093/nar/11.11.3823. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Tadaki T., Fukushima M., Ushida C., Himeno H., Muto A. Interaction of 10Sa RNA with ribosomes in Escherichia coli. FEBS Lett. 1996 Dec 16;399(3):223–226. doi: 10.1016/s0014-5793(96)01330-0. [DOI] [PubMed] [Google Scholar]
  34. Tate W. P., Brown C. M. Translational termination: "stop" for protein synthesis or "pause" for regulation of gene expression. Biochemistry. 1992 Mar 10;31(9):2443–2450. doi: 10.1021/bi00124a001. [DOI] [PubMed] [Google Scholar]
  35. Tate W. P., Poole E. S., Dalphin M. E., Major L. L., Crawford D. J., Mannering S. A. The translational stop signal: codon with a context, or extended factor recognition element? Biochimie. 1996;78(11-12):945–952. doi: 10.1016/s0300-9084(97)86716-8. [DOI] [PubMed] [Google Scholar]
  36. Thompson R. C. EFTu provides an internal kinetic standard for translational accuracy. Trends Biochem Sci. 1988 Mar;13(3):91–93. doi: 10.1016/0968-0004(88)90047-3. [DOI] [PubMed] [Google Scholar]
  37. 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]
  38. Vind J., Sørensen M. A., Rasmussen M. D., Pedersen S. Synthesis of proteins in Escherichia coli is limited by the concentration of free ribosomes. Expression from reporter genes does not always reflect functional mRNA levels. J Mol Biol. 1993 Jun 5;231(3):678–688. doi: 10.1006/jmbi.1993.1319. [DOI] [PubMed] [Google Scholar]
  39. Weiss R. B., Dunn D. M., Atkins J. F., Gesteland R. F. Slippery runs, shifty stops, backward steps, and forward hops: -2, -1, +1, +2, +5, and +6 ribosomal frameshifting. Cold Spring Harb Symp Quant Biol. 1987;52:687–693. doi: 10.1101/sqb.1987.052.01.078. [DOI] [PubMed] [Google Scholar]
  40. Weiss R. B., Dunn D. M., Dahlberg A. E., Atkins J. F., Gesteland R. F. Reading frame switch caused by base-pair formation between the 3' end of 16S rRNA and the mRNA during elongation of protein synthesis in Escherichia coli. EMBO J. 1988 May;7(5):1503–1507. doi: 10.1002/j.1460-2075.1988.tb02969.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Zhang S., Rydén-Aulin M., Isaksson L. A. Functional interaction between release factor one and P-site peptidyl-tRNA on the ribosome. J Mol Biol. 1996 Aug 16;261(2):98–107. doi: 10.1006/jmbi.1996.0444. [DOI] [PubMed] [Google Scholar]
  42. de Smit M. H., van Duin J., van Knippenberg P. H., van Eijk H. G. CCC.UGA: a new site of ribosomal frameshifting in Escherichia coli. Gene. 1994 May 27;143(1):43–47. doi: 10.1016/0378-1119(94)90602-5. [DOI] [PubMed] [Google Scholar]

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