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. 2000 Jul;6(7):1044–1055. doi: 10.1017/s1355838200000716

Aminoglycoside antibiotics mediate context-dependent suppression of termination codons in a mammalian translation system.

M Manuvakhova 1, K Keeling 1, D M Bedwell 1
PMCID: PMC1369979  PMID: 10917599

Abstract

The translation machinery recognizes codons that enter the ribosomal A site with remarkable accuracy to ensure that polypeptide synthesis proceeds with a minimum of errors. When a termination codon enters the A site of a eukaryotic ribosome, it is recognized by the release factor eRF1. It has been suggested that the recognition of translation termination signals in these organisms is not limited to a simple trinucleotide codon, but is instead recognized by an extended tetranucleotide termination signal comprised of the stop codon and the first nucleotide that follows. Interestingly, pharmacological agents such as aminoglycoside antibiotics can reduce the efficiency of translation termination by a mechanism that alters this ribosomal proofreading process. This leads to the misincorporation of an amino acid through the pairing of a near-cognate aminoacyl tRNA with the stop codon. To determine whether the sequence context surrounding a stop codon can influence aminoglycoside-mediated suppression of translation termination signals, we developed a series of readthrough constructs that contained different tetranucleotide termination signals, as well as differences in the three bases upstream and downstream of the stop codon. Our results demonstrate that the sequences surrounding a stop codon can play an important role in determining its susceptibility to suppression by aminoglycosides. Furthermore, these distal sequences were found to influence the level of suppression in remarkably distinct ways. These results suggest that the mRNA context influences the suppression of stop codons in response to subtle differences in the conformation of the ribosomal decoding site that result from aminoglycoside binding.

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Selected References

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

  1. Atkinson J., Martin R. Mutations to nonsense codons in human genetic disease: implications for gene therapy by nonsense suppressor tRNAs. Nucleic Acids Res. 1994 Apr 25;22(8):1327–1334. doi: 10.1093/nar/22.8.1327. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Barton-Davis E. R., Cordier L., Shoturma D. I., Leland S. E., Sweeney H. L. Aminoglycoside antibiotics restore dystrophin function to skeletal muscles of mdx mice. J Clin Invest. 1999 Aug;104(4):375–381. doi: 10.1172/JCI7866. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bedwell D. M., Kaenjak A., Benos D. J., Bebok Z., Bubien J. K., Hong J., Tousson A., Clancy J. P., Sorscher E. J. Suppression of a CFTR premature stop mutation in a bronchial epithelial cell line. Nat Med. 1997 Nov;3(11):1280–1284. doi: 10.1038/nm1197-1280. [DOI] [PubMed] [Google Scholar]
  4. Bonetti B., Fu L., Moon J., Bedwell D. M. The efficiency of translation termination is determined by a synergistic interplay between upstream and downstream sequences in Saccharomyces cerevisiae. J Mol Biol. 1995 Aug 18;251(3):334–345. doi: 10.1006/jmbi.1995.0438. [DOI] [PubMed] [Google Scholar]
  5. Bossi L., Ruth J. R. The influence of codon context on genetic code translation. Nature. 1980 Jul 10;286(5769):123–127. doi: 10.1038/286123a0. [DOI] [PubMed] [Google Scholar]
  6. Brown C. M., Dalphin M. E., Stockwell P. A., Tate W. P. The translational termination signal database. Nucleic Acids Res. 1993 Jul 1;21(13):3119–3123. doi: 10.1093/nar/21.13.3119. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Brown C. M., Stockwell P. A., Trotman C. N., Tate W. P. Sequence analysis suggests that tetra-nucleotides signal the termination of protein synthesis in eukaryotes. Nucleic Acids Res. 1990 Nov 11;18(21):6339–6345. doi: 10.1093/nar/18.21.6339. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Buckingham R. H., Grentzmann G., Kisselev L. Polypeptide chain release factors. Mol Microbiol. 1997 May;24(3):449–456. doi: 10.1046/j.1365-2958.1997.3711734.x. [DOI] [PubMed] [Google Scholar]
  9. Cate J. H., Yusupov M. M., Yusupova G. Z., Earnest T. N., Noller H. F. X-ray crystal structures of 70S ribosome functional complexes. Science. 1999 Sep 24;285(5436):2095–2104. doi: 10.1126/science.285.5436.2095. [DOI] [PubMed] [Google Scholar]
  10. Czaplinski K., Ruiz-Echevarria M. J., González C. I., Peltz S. W. Should we kill the messenger? The role of the surveillance complex in translation termination and mRNA turnover. Bioessays. 1999 Aug;21(8):685–696. doi: 10.1002/(SICI)1521-1878(199908)21:8<685::AID-BIES8>3.0.CO;2-4. [DOI] [PubMed] [Google Scholar]
  11. Czaplinski K., Ruiz-Echevarria M. J., Paushkin S. V., Han X., Weng Y., Perlick H. A., Dietz H. C., Ter-Avanesyan M. D., Peltz S. W. The surveillance complex interacts with the translation release factors to enhance termination and degrade aberrant mRNAs. Genes Dev. 1998 Jun 1;12(11):1665–1677. doi: 10.1101/gad.12.11.1665. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Fearon K., McClendon V., Bonetti B., Bedwell D. M. Premature translation termination mutations are efficiently suppressed in a highly conserved region of yeast Ste6p, a member of the ATP-binding cassette (ABC) transporter family. J Biol Chem. 1994 Jul 8;269(27):17802–17808. [PubMed] [Google Scholar]
  13. Feinstein S. I., Altman S. Coding properties of an ochre-suppressing derivative of Escherichia coli tRNAITyr. J Mol Biol. 1977 May 25;112(3):453–470. doi: 10.1016/s0022-2836(77)80192-7. [DOI] [PubMed] [Google Scholar]
  14. Feng Y. X., Yuan H., Rein A., Levin J. G. Bipartite signal for read-through suppression in murine leukemia virus mRNA: an eight-nucleotide purine-rich sequence immediately downstream of the gag termination codon followed by an RNA pseudoknot. J Virol. 1992 Aug;66(8):5127–5132. doi: 10.1128/jvi.66.8.5127-5132.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Fourmy D., Recht M. I., Blanchard S. C., Puglisi J. D. Structure of the A site of Escherichia coli 16S ribosomal RNA complexed with an aminoglycoside antibiotic. Science. 1996 Nov 22;274(5291):1367–1371. doi: 10.1126/science.274.5291.1367. [DOI] [PubMed] [Google Scholar]
  16. Freistroffer D. V., Pavlov M. Y., MacDougall J., Buckingham R. H., Ehrenberg M. Release factor RF3 in E.coli accelerates the dissociation of release factors RF1 and RF2 from the ribosome in a GTP-dependent manner. EMBO J. 1997 Jul 1;16(13):4126–4133. doi: 10.1093/emboj/16.13.4126. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. 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]
  18. Gesteland R. F., Weiss R. B., Atkins J. F. Recoding: reprogrammed genetic decoding. Science. 1992 Sep 18;257(5077):1640–1641. doi: 10.1126/science.1529352. [DOI] [PubMed] [Google Scholar]
  19. Howard M., Frizzell R. A., Bedwell D. M. Aminoglycoside antibiotics restore CFTR function by overcoming premature stop mutations. Nat Med. 1996 Apr;2(4):467–469. doi: 10.1038/nm0496-467. [DOI] [PubMed] [Google Scholar]
  20. 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]
  21. Lodmell J. S., Dahlberg A. E. A conformational switch in Escherichia coli 16S ribosomal RNA during decoding of messenger RNA. Science. 1997 Aug 29;277(5330):1262–1267. doi: 10.1126/science.277.5330.1262. [DOI] [PubMed] [Google Scholar]
  22. Martin R., Mogg A. E., Heywood L. A., Nitschke L., Burke J. F. Aminoglycoside suppression at UAG, UAA and UGA codons in Escherichia coli and human tissue culture cells. Mol Gen Genet. 1989 Jun;217(2-3):411–418. doi: 10.1007/BF02464911. [DOI] [PubMed] [Google Scholar]
  23. 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]
  24. Milman G., Goldstein J., Scolnick E., Caskey T. Peptide chain termination. 3. Stimulation of in vitro termination. Proc Natl Acad Sci U S A. 1969 May;63(1):183–190. doi: 10.1073/pnas.63.1.183. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Moffat J. G., Tate W. P., Lovett P. S. The leader peptides of attenuation-regulated chloramphenicol resistance genes inhibit translational termination. J Bacteriol. 1994 Nov;176(22):7115–7117. doi: 10.1128/jb.176.22.7115-7117.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. 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]
  27. 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]
  28. Pape T., Wintermeyer W., Rodnina M. V. Conformational switch in the decoding region of 16S rRNA during aminoacyl-tRNA selection on the ribosome. Nat Struct Biol. 2000 Feb;7(2):104–107. doi: 10.1038/72364. [DOI] [PubMed] [Google Scholar]
  29. Pape T., Wintermeyer W., Rodnina M. Induced fit in initial selection and proofreading of aminoacyl-tRNA on the ribosome. EMBO J. 1999 Jul 1;18(13):3800–3807. doi: 10.1093/emboj/18.13.3800. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. 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]
  31. Phillips-Jones M. K., Hill L. S., Atkinson J., Martin R. Context effects on misreading and suppression at UAG codons in human cells. Mol Cell Biol. 1995 Dec;15(12):6593–6600. doi: 10.1128/mcb.15.12.6593. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. 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]
  33. Poole E. S., Major L. L., Mannering S. A., Tate W. P. Translational termination in Escherichia coli: three bases following the stop codon crosslink to release factor 2 and affect the decoding efficiency of UGA-containing signals. Nucleic Acids Res. 1998 Feb 15;26(4):954–960. doi: 10.1093/nar/26.4.954. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Purohit P., Stern S. Interactions of a small RNA with antibiotic and RNA ligands of the 30S subunit. Nature. 1994 Aug 25;370(6491):659–662. doi: 10.1038/370659a0. [DOI] [PubMed] [Google Scholar]
  35. Recht M. I., Douthwaite S., Puglisi J. D. Basis for prokaryotic specificity of action of aminoglycoside antibiotics. EMBO J. 1999 Jun 1;18(11):3133–3138. doi: 10.1093/emboj/18.11.3133. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Recht M. I., Fourmy D., Blanchard S. C., Dahlquist K. D., Puglisi J. D. RNA sequence determinants for aminoglycoside binding to an A-site rRNA model oligonucleotide. J Mol Biol. 1996 Oct 4;262(4):421–436. doi: 10.1006/jmbi.1996.0526. [DOI] [PubMed] [Google Scholar]
  37. Rivera A. A., Elton T. S., Dey N. B., Bounelis P., Marchase R. B. Isolation and expression of a rat liver cDNA encoding phosphoglucomutase. Gene. 1993 Nov 15;133(2):261–266. doi: 10.1016/0378-1119(93)90649-n. [DOI] [PubMed] [Google Scholar]
  38. Scolnick E., Tompkins R., Caskey T., Nirenberg M. Release factors differing in specificity for terminator codons. Proc Natl Acad Sci U S A. 1968 Oct;61(2):768–774. doi: 10.1073/pnas.61.2.768. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. 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]
  40. Wills N. M., Gesteland R. F., Atkins J. F. Evidence that a downstream pseudoknot is required for translational read-through of the Moloney murine leukemia virus gag stop codon. Proc Natl Acad Sci U S A. 1991 Aug 15;88(16):6991–6995. doi: 10.1073/pnas.88.16.6991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Yoshinaka Y., Katoh I., Copeland T. D., Oroszlan S. Murine leukemia virus protease is encoded by the gag-pol gene and is synthesized through suppression of an amber termination codon. Proc Natl Acad Sci U S A. 1985 Mar;82(6):1618–1622. doi: 10.1073/pnas.82.6.1618. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Yoshizawa S., Fourmy D., Puglisi J. D. Recognition of the codon-anticodon helix by ribosomal RNA. Science. 1999 Sep 10;285(5434):1722–1725. doi: 10.1126/science.285.5434.1722. [DOI] [PubMed] [Google Scholar]
  43. Yoshizawa S., Fourmy D., Puglisi J. D. Structural origins of gentamicin antibiotic action. EMBO J. 1998 Nov 16;17(22):6437–6448. doi: 10.1093/emboj/17.22.6437. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. 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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