Skip to main content
PMC home page
RNA logoLink to RNA
. 2000 Aug;6(8):1174–1184. doi: 10.1017/s1355838200000637

Mutations in helix 27 of the yeast Saccharomyces cerevisiae 18S rRNA affect the function of the decoding center of the ribosome.

I V Velichutina 1, J Dresios 1, J Y Hong 1, C Li 1, A Mankin 1, D Synetos 1, S W Liebman 1
PMCID: PMC1369991  PMID: 10943896

Abstract

A dynamic structural rearrangement in the phylogenetically conserved helix 27 of Escherichia coli 16S rRNA has been proposed to directly affect the accuracy of translational decoding by switching between "accurate" and "error-prone" conformations. To examine the function of helix 27 in eukaryotes, random and site-specific mutations in helix 27 of the yeast Saccharomyces cerevisiae 18S rRNA have been characterized. Mutations at positions of yeast 18S rRNA corresponding to E. coli 886 (rdn8), 888 (rdn6), and 912 (rdn4) increased translational accuracy in vivo and in vitro, and caused a reduction in tRNA binding to the A-site of mutant ribosomes. The double rdn4rdn6 mutation separated the killing and stop-codon readthrough effects of the aminoglycoside antibiotic, paromomycin, implicating a direct involvement of yeast helix 27 in accurate recognition of codons by tRNA or release factor eRF1. Although our data in yeast does not support a conformational switch model analogous to that proposed for helix 27 of E. coli 16S rRNA, it strongly suggests a functional conservation of this region in tRNA selection.

Full Text

The Full Text of this article is available as a PDF (754.0 KB).

Selected References

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

  1. Arkov A. L., Freistroffer D. V., Ehrenberg M., Murgola E. J. Mutations in RNAs of both ribosomal subunits cause defects in translation termination. EMBO J. 1998 Mar 2;17(5):1507–1514. doi: 10.1093/emboj/17.5.1507. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Boeke J. D., LaCroute F., Fink G. R. A positive selection for mutants lacking orotidine-5'-phosphate decarboxylase activity in yeast: 5-fluoro-orotic acid resistance. Mol Gen Genet. 1984;197(2):345–346. doi: 10.1007/BF00330984. [DOI] [PubMed] [Google Scholar]
  3. 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]
  4. Chernoff Y. O., Newnam G. P., Liebman S. W. The translational function of nucleotide C1054 in the small subunit rRNA is conserved throughout evolution: genetic evidence in yeast. Proc Natl Acad Sci U S A. 1996 Mar 19;93(6):2517–2522. doi: 10.1073/pnas.93.6.2517. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Chernoff Y. O., Vincent A., Liebman S. W. Mutations in eukaryotic 18S ribosomal RNA affect translational fidelity and resistance to aminoglycoside antibiotics. EMBO J. 1994 Feb 15;13(4):906–913. doi: 10.1002/j.1460-2075.1994.tb06334.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Clemons W. M., Jr, May J. L., Wimberly B. T., McCutcheon J. P., Capel M. S., Ramakrishnan V. Structure of a bacterial 30S ribosomal subunit at 5.5 A resolution. Nature. 1999 Aug 26;400(6747):833–840. doi: 10.1038/23631. [DOI] [PubMed] [Google Scholar]
  7. Coutsogeorgopoulos C., Miller J. T., Hann D. M. Inhibitors of protein synthesis V. Irreversible interaction of antibiotics with an initiation complex. Nucleic Acids Res. 1975 Jul;2(7):1053–1072. doi: 10.1093/nar/2.7.1053. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Fourmy D., Yoshizawa S., Puglisi J. D. Paromomycin binding induces a local conformational change in the A-site of 16 S rRNA. J Mol Biol. 1998 Mar 27;277(2):333–345. doi: 10.1006/jmbi.1997.1551. [DOI] [PubMed] [Google Scholar]
  9. Gabashvili I. S., Agrawal R. K., Grassucci R., Squires C. L., Dahlberg A. E., Frank J. Major rearrangements in the 70S ribosomal 3D structure caused by a conformational switch in 16S ribosomal RNA. EMBO J. 1999 Nov 15;18(22):6501–6507. doi: 10.1093/emboj/18.22.6501. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Grant P., Sánchez L., Jiménez A. Cryptopleurine resistance: genetic locus for a 40S ribosomal component in Saccharomyces cerevisiae. J Bacteriol. 1974 Dec;120(3):1308–1314. doi: 10.1128/jb.120.3.1308-1314.1974. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Gutell R. R., Larsen N., Woese C. R. Lessons from an evolving rRNA: 16S and 23S rRNA structures from a comparative perspective. Microbiol Rev. 1994 Mar;58(1):10–26. doi: 10.1128/mr.58.1.10-26.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. 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]
  13. Hussain I., Leibowitz M. J. Translation of homologous and heterologous messenger RNAs in a yeast cell-free system. Gene. 1986;46(1):13–23. doi: 10.1016/0378-1119(86)90162-9. [DOI] [PubMed] [Google Scholar]
  14. Ito H., Fukuda Y., Murata K., Kimura A. Transformation of intact yeast cells treated with alkali cations. J Bacteriol. 1983 Jan;153(1):163–168. doi: 10.1128/jb.153.1.163-168.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Ito W., Ishiguro H., Kurosawa Y. A general method for introducing a series of mutations into cloned DNA using the polymerase chain reaction. Gene. 1991 Jun 15;102(1):67–70. doi: 10.1016/0378-1119(91)90539-n. [DOI] [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. Kraal B., Zeef L. A., Mesters J. R., Boon K., Vorstenbosch E. L., Bosch L., Anborgh P. H., Parmeggiani A., Hilgenfeld R. Antibiotic resistance mechanisms of mutant EF-Tu species in Escherichia coli. Biochem Cell Biol. 1995 Nov-Dec;73(11-12):1167–1177. doi: 10.1139/o95-126. [DOI] [PubMed] [Google Scholar]
  18. Kurland C. G. Translational accuracy and the fitness of bacteria. Annu Rev Genet. 1992;26:29–50. doi: 10.1146/annurev.ge.26.120192.000333. [DOI] [PubMed] [Google Scholar]
  19. Leibowitz M. J., Barbone F. P., Georgopoulos D. E. In vitro protein synthesis. Methods Enzymol. 1991;194:536–545. doi: 10.1016/0076-6879(91)94040-j. [DOI] [PubMed] [Google Scholar]
  20. Liebman S. W., Chernoff Y. O., Liu R. The accuracy center of a eukaryotic ribosome. Biochem Cell Biol. 1995 Nov-Dec;73(11-12):1141–1149. doi: 10.1139/o95-123. [DOI] [PubMed] [Google Scholar]
  21. Lill R., Robertson J. M., Wintermeyer W. tRNA binding sites of ribosomes from Escherichia coli. Biochemistry. 1984 Dec 18;23(26):6710–6717. doi: 10.1021/bi00321a066. [DOI] [PubMed] [Google Scholar]
  22. Liu R., Liebman S. W. A translational fidelity mutation in the universally conserved sarcin/ricin domain of 25S yeast ribosomal RNA. RNA. 1996 Mar;2(3):254–263. [PMC free article] [PubMed] [Google Scholar]
  23. 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]
  24. Lodmell J. S., Gutell R. R., Dahlberg A. E. Genetic and comparative analyses reveal an alternative secondary structure in the region of nt 912 of Escherichia coli 16S rRNA. Proc Natl Acad Sci U S A. 1995 Nov 7;92(23):10555–10559. doi: 10.1073/pnas.92.23.10555. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Mathews D. H., Sabina J., Zuker M., Turner D. H. Expanded sequence dependence of thermodynamic parameters improves prediction of RNA secondary structure. J Mol Biol. 1999 May 21;288(5):911–940. doi: 10.1006/jmbi.1999.2700. [DOI] [PubMed] [Google Scholar]
  26. Merryman C., Moazed D., McWhirter J., Noller H. F. Nucleotides in 16S rRNA protected by the association of 30S and 50S ribosomal subunits. J Mol Biol. 1999 Jan 8;285(1):97–105. doi: 10.1006/jmbi.1998.2242. [DOI] [PubMed] [Google Scholar]
  27. Moazed D., Noller H. F. Interaction of antibiotics with functional sites in 16S ribosomal RNA. Nature. 1987 Jun 4;327(6121):389–394. doi: 10.1038/327389a0. [DOI] [PubMed] [Google Scholar]
  28. Moazed D., Noller H. F. Transfer RNA shields specific nucleotides in 16S ribosomal RNA from attack by chemical probes. Cell. 1986 Dec 26;47(6):985–994. doi: 10.1016/0092-8674(86)90813-5. [DOI] [PubMed] [Google Scholar]
  29. Mueller F., Brimacombe R. A new model for the three-dimensional folding of Escherichia coli 16 S ribosomal RNA. I. Fitting the RNA to a 3D electron microscopic map at 20 A. J Mol Biol. 1997 Aug 29;271(4):524–544. doi: 10.1006/jmbi.1997.1210. [DOI] [PubMed] [Google Scholar]
  30. Mugnier P., Tuite M. F. Translation termination and its regulation in eukaryotes: recent insights provided by studies in yeast. Biochemistry (Mosc) 1999 Dec;64(12):1360–1366. [PubMed] [Google Scholar]
  31. O'Connor M., Göringer H. U., Dahlberg A. E. A ribosomal ambiguity mutation in the 530 loop of E. coli 16S rRNA. Nucleic Acids Res. 1992 Aug 25;20(16):4221–4227. doi: 10.1093/nar/20.16.4221. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. O'Connor M., Thomas C. L., Zimmermann R. A., Dahlberg A. E. Decoding fidelity at the ribosomal A and P sites: influence of mutations in three different regions of the decoding domain in 16S rRNA. Nucleic Acids Res. 1997 Mar 15;25(6):1185–1193. doi: 10.1093/nar/25.6.1185. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Pagel F. T., Zhao S. Q., Hijazi K. A., Murgola E. J. Phenotypic heterogeneity of mutational changes at a conserved nucleotide in 16 S ribosomal RNA. J Mol Biol. 1997 Apr 18;267(5):1113–1123. doi: 10.1006/jmbi.1997.0943. [DOI] [PubMed] [Google Scholar]
  34. Palmer E., Wilhelm J. M., Sherman F. Phenotypic suppression of nonsense mutants in yeast by aminoglycoside antibiotics. Nature. 1979 Jan 11;277(5692):148–150. doi: 10.1038/277148a0. [DOI] [PubMed] [Google Scholar]
  35. 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]
  36. Parent S. A., Fenimore C. M., Bostian K. A. Vector systems for the expression, analysis and cloning of DNA sequences in S. cerevisiae. Yeast. 1985 Dec;1(2):83–138. doi: 10.1002/yea.320010202. [DOI] [PubMed] [Google Scholar]
  37. Ruusala T., Ehrenberg M., Kurland C. G. Is there proofreading during polypeptide synthesis? EMBO J. 1982;1(6):741–745. doi: 10.1002/j.1460-2075.1982.tb01240.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Song H., Mugnier P., Das A. K., Webb H. M., Evans D. R., Tuite M. F., Hemmings B. A., Barford D. The crystal structure of human eukaryotic release factor eRF1--mechanism of stop codon recognition and peptidyl-tRNA hydrolysis. Cell. 2000 Feb 4;100(3):311–321. doi: 10.1016/s0092-8674(00)80667-4. [DOI] [PubMed] [Google Scholar]
  39. Stern S., Powers T., Changchien L. M., Noller H. F. RNA-protein interactions in 30S ribosomal subunits: folding and function of 16S rRNA. Science. 1989 May 19;244(4906):783–790. doi: 10.1126/science.2658053. [DOI] [PubMed] [Google Scholar]
  40. Stern S., Wilson R. C., Noller H. F. Localization of the binding site for protein S4 on 16 S ribosomal RNA by chemical and enzymatic probing and primer extension. J Mol Biol. 1986 Nov 5;192(1):101–110. doi: 10.1016/0022-2836(86)90467-5. [DOI] [PubMed] [Google Scholar]
  41. Synetos D., Coutsogeorgopoulos C. Studies on the catalytic rate constant of ribosomal peptidyltransferase. Biochim Biophys Acta. 1987 Feb 20;923(2):275–285. doi: 10.1016/0304-4165(87)90014-6. [DOI] [PubMed] [Google Scholar]
  42. Synetos D., Frantziou C. P., Alksne L. E. Mutations in yeast ribosomal proteins S28 and S4 affect the accuracy of translation and alter the sensitivity of the ribosomes to paromomycin. Biochim Biophys Acta. 1996 Nov 11;1309(1-2):156–166. doi: 10.1016/s0167-4781(96)00128-5. [DOI] [PubMed] [Google Scholar]
  43. Thompson R. C., Dix D. B., Karim A. M. The reaction of ribosomes with elongation factor Tu.GTP complexes. Aminoacyl-tRNA-independent reactions in the elongation cycle determine the accuracy of protein synthesis. J Biol Chem. 1986 Apr 15;261(11):4868–4874. [PubMed] [Google Scholar]
  44. VanLoock M. S., Easterwood T. R., Harvey S. C. Major groove binding of the tRNA/mRNA complex to the 16 S ribosomal RNA decoding site. J Mol Biol. 1999 Feb 5;285(5):2069–2078. doi: 10.1006/jmbi.1998.2442. [DOI] [PubMed] [Google Scholar]
  45. Vijgenboom E., Vink T., Kraal B., Bosch L. Mutants of the elongation factor EF-Tu, a new class of nonsense suppressors. EMBO J. 1985 Apr;4(4):1049–1052. doi: 10.1002/j.1460-2075.1985.tb03737.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Wilson K. S., Noller H. F. Molecular movement inside the translational engine. Cell. 1998 Feb 6;92(3):337–349. doi: 10.1016/s0092-8674(00)80927-7. [DOI] [PubMed] [Google Scholar]
  47. 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]
  48. Zuker M., Jacobson A. B. Using reliability information to annotate RNA secondary structures. RNA. 1998 Jun;4(6):669–679. doi: 10.1017/s1355838298980116. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from RNA are provided here courtesy of The RNA Society

RESOURCES