Current Genetics

, Volume 27, Issue 5, pp 417–426 | Cite as

A mutant allele of the SUP45 (SAL4) gene of Saccharomyces cerevisiae shows temperature-dependent allosuppressor and omnipotent suppressor phenotypes

  • I. Stansfield
  • Akhmaloka
  • M. F. Tuite
Original Paper

Abstract

Using a plasmid-based termination-read-through assay, the sal4-2 conditional-lethal (temperature-sensitive) allele of the SUP45 (SAL4) gene was shown to enhance the efficiency of the weak ochre suppressor tRNA SUQ5 some 10-fold at 30°C. Additionally, this allele increased the suppressor efficiency of SRM2-2, a weak tRNAGln ochre suppressor, indicating that the allosuppressor phenotype is not SUQ5-specific. A sup+ sal4-2 strain also showed a temperature-dependent omnipotent suppressor phenotype, enhancing readthrough of all three termination codons. Combining the sal4-2 allele with an efficient tRNA nonsense suppressor (SUP4) increased the temperature-sensitivity of that strain, indicating that enhanced nonsense suppressor levels contribute to the conditional-lethality conferred by the sal4-2 allele. However, UGA suppression levels in a sup+ sal4-2 strain following a shift to the non-permissive temperature reached a maximum significantly below that exhibited by a non-temperature sensitive SUP4 suppressor strain. Enhanced nonsense suppression may not therefore be the primary cause of the conditional-lethality of this allele. These data indicate a role for Sup45p in translation termination, and possibly in an additional, as yet unidentified, cellular process.

Key words

Saccharomyces cerevisiae Omnipotent suppression Nonsense suppression SUP45 

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

References

  1. Akhmaloka (1991) A molecular genetic analysis of the allosuppressor gene SAL4 in Saccharomyces cerevisiae. PhD thesis, University of Kent, CanterburyGoogle Scholar
  2. All-Robyn JA, Brown N, Otaka E, Liebman SW (1990) Sequence and functional similarity between a yeast ribosomal protein and the Escherichia coli S5 ram protein. Mol Cell Biol 10:6544–6553Google Scholar
  3. Boone C, Clark KL, Sprague GF (1992) Identification of a tRNAGln ochre suppressor in Saccharomyces cerevisiae. Nucleic Acids Res 20:4661Google Scholar
  4. Breining P, Piepersberg W (1986) Yeast omnipotent suppressor SUP1 (SUP45): nucleotide sequence of the wild-type and a mutant gene. Nucleic Acids Res 14:5187–5197Google Scholar
  5. Brown CM, Stockwell PA, Trotman CNA, Tate WP (1990) Sequence analysis suggests that tetranucleotides signal the termination of protein synthesis in eukaryotes. Nucleic Acids Res 18:6339–6345Google Scholar
  6. Christianson TW, Sikorski RS, Dante M, Shero JH, Heiter P (1992) Multifunctional yeast high-copy number shuttle vectors. Gene 110:119–122Google Scholar
  7. Cox BS (1977) Allosuppressors in yeast. Genet Res 30:187–205Google Scholar
  8. Crouzet M, Igzu F, Grant CM, Tuite MF (1988) The allosuppressor gene SAL4 encodes a protein important for maintaining translational fidelity in Saccharomyces cerevisiae. Curr Genet 14:537–543Google Scholar
  9. Davidoff-Abelson R, Mindich L (1978) A mutation that increases the activity of nonsense suppressors in Escherichia coli. Mol Gen Genet 159:161–169Google Scholar
  10. Devereaux J, Haeberli P, Smithies O (1984) A comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Res 12:387–395Google Scholar
  11. Didichenko SA, Ter-Avanesyan MD, Smirnov VN (1991) Ribosomebound EF1-α-like protein of yeast Saccharomyces cerevisiae. Eur J Biochem 198:705–711Google Scholar
  12. Eustice DC, Wakem LP, Wilhelm JM, Sherman F (1986) Altered 40 S ribosomal subunits in omnipotent suppressors of yeast. J Mol Biol 188:207–214Google Scholar
  13. Firoozan M, Grant CM, Duarte JAB, Tuite MF (1991) Quantitation of readthrough of termination codons in yeast using a novel gene fusion assay. Yeast 7:173–184Google Scholar
  14. Finkelstein DB, Strausberg S (1983) Heat shock-regulated production of E. coli β-galactosidase in Saccharomyces cerevisiae. Mol Cell Biol 3:1625–1633Google Scholar
  15. Goodman HM, Olson MV, Hall BD (1977) Nucleotide sequence of a mutant eukaryotic gene: the yeast tyrosine-inserting ochre-suppressor SUP4-o. Proc Natl Acad Sci USA 74:5453–5457Google Scholar
  16. Hawthorne DC, Leupold U (1974) Suppressor mutations in yeast. Curr Top Microbiol Immunol 64:1–47Google Scholar
  17. Hoffman CS, Winston F (1987) A ten-minute DNA preparation from yeast efficiently releases autonomous plasmids for transformation of Escherichia coli. Gene 57:267–272Google Scholar
  18. Inge-Vechtomov SG, Andrianova VM (1970) Recessive super suppressors in yeast Genetika 6:103–115Google Scholar
  19. Ito H, Wittman HG (1973) Amino-acid replacements in proteins S5 and S12 of two Escherichia coli revertants from streptomycin dependence to independence. Mol Gen Genet 127:19–32Google Scholar
  20. Ito M, Fukada Y, Murata K, Kimura A (1983) Transformation of intact yeast cells treated with alkali cations. J Bacteriol 153:163–168Google Scholar
  21. Konecki DS, Aune KC, Tate W, Caskey CT (1977) Characterisation of reticulocyte release factor. J Biol Chem 252:4514–4520Google Scholar
  22. Laten H, Gorman J, Bock RM (1978) Isopentenyladenosine-deficient tRNA from an antisuppressor mutant of Saccharomyces cerevisiae. Nucleic Acids Res 5:4329–4342Google Scholar
  23. Leeds P, Wood JM, Lee B-S, Culbertson MR (1992) Gene products that promote mRNA turnover in Saccharomyces cerevisiae. Mol Cell Biol 12:2165–2177Google Scholar
  24. Liebman SW, Sherman F (1976) Inhibition of growth by amber suppressors in yeast. Genetics 82:233–249Google Scholar
  25. Manivasakam P, Schiest RH (1993) High-efficiency transformation of Saccharomyces cerevisiae by electroporation. Nucleic Acids Res 21:4414–4415Google Scholar
  26. Milman G, Goldstein J, Scolnick E, Caskey CT (1969) Peptide termination. III. stimulation of in vitro termination, Proc Natl Acad Sci USA 63:183–190Google Scholar
  27. Mikuni O, Ito K, Moffat J, Matsumara K, McCaughan K, Nobukuni T, Tate W, Nakamura Y (1994) Identification of the prfC gene, which encodes peptide chain release factor-3 of Escherichia coli. Proc Natl Acad Sci USA 91:5798–5802Google Scholar
  28. Orr-Weaver TL, Szostak JW (1983) Yeast recombination: the association between double-strand gap repair and crossing over. Proc Natl Acad Sci USA 80:4417–4420Google Scholar
  29. Pure GA, Robinson GW, Naumovski L, Frieberg EC (1988) Partial suppression of an ochre mutation in Saccharomyces cerevisiae by multicopy plasmids containing a normal tRNAgln gene. J Mol Biol 183:31–42Google Scholar
  30. Rose MD, Novick P, Thomas JH, Botstein D, Fink GR (1987) A Saccharomyces cerevisiae genomic plasmid bank based on a centromere-containing shuttle vector. Gene 60:237–243Google Scholar
  31. Rosset R, Gorini L (1969) A ribosomal ambiguity mutation. J Mol Biol 39:95–107Google Scholar
  32. Ryden SM, Isaksson LA (1984) 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 193:38–45Google Scholar
  33. Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual, 2nd edn. Cold Spring Harbour Laboratory, Cold Spring Harbour, New YorkGoogle Scholar
  34. Sanger F, Nicklen S, Coulson AR (1977) DNA sequencing with chaintermination inhibitors. Proc Natl Acad Sci USA 74:5463–5469Google Scholar
  35. Sherman F (1982) Suppression in the yeast Saccharomyces cerevisiae. In: Strathern J, Jones E, Broach J (eds) The molecular biology of the yeast Sacharomyces: metabolism and gene expression. Cold Spring Harbour Laboratory, Cold Spring Harbour, New York, pp 463–486Google Scholar
  36. Sherman F (1991) Getting started with yeast. Methods Enzymol 194:3–20Google Scholar
  37. Sherman F, Hicks J (1991) Micromanipulation and dissection of asci. Methods Enzymol 194:21–37Google Scholar
  38. Slobin LI (1980) The role of eukaryotic elongation factor Tu in protein synthesis. Eur J Biochem 110:555–563Google Scholar
  39. Stansfield I, Tuite MF (1994) Polypeptide chain-termination in Saccharomyces cerevisiae. Curr Genet 25:385–395Google Scholar
  40. Stansfield I, Grant CM, Akhmaloka, Tuite MF (1992) Ribosomal association of the yeast SAL4(SUP45) gene product: implications for its role in translation fidelity and termination. Mol Microbiol 16:3469–3478Google Scholar
  41. Surguchov AP, Smirnov VN, Ter-Avanesyan MD, Inge-Vechtomov SG (1984) Ribosomal suppression in eukaryotes. Phys Chem Biol Rev 4:147–205Google Scholar
  42. Tuite MF, McLaughlin CS (1982) Natural readthrough of a UGA termination codon in a yeast cell-free system: evidence for involvement of both a mitochondrial and nuclear tRNA. Mol Cell Biol 2:490–497Google Scholar
  43. Tuite MF, Akhmaloka, Firoozan M, Duarte JAB, Grant CM (1990) Controlling translational accuracy in Saccharomyces cerevisiae: the molecular genetic analysis of a key translation factor, the Sal4 protein. In: McCarthy JEG, Tuite MF (eds) Post-transcriptional control of gene-expression. Springer, Berlin, pp 611–622Google Scholar
  44. Vincent A, Liebman SW (1992) The yeast omnipotent suppressor SUP46 encodes a ribosomal protein which is a functional and structural homolog of the Escherichia coli S4 ram protein. Genetics 132:375–386Google Scholar
  45. Waldron C, Cox BS, Wills N, Gestland RF, Piper PW, Colby D, Guthrie C (1981) Yeast ochre suppressor SUQ5-o 1 is an altered tRNAserUCA. Nucleic Acids Res 9:3077–3088Google Scholar
  46. Weiss WA, Friedberg EC (1987) Normal yeast tRNAglnCAG can suppress amber codons and is encoded by an essential gene. J Mol Biol 192:725–735Google Scholar

Copyright information

© Springer-Verlag 1995

Authors and Affiliations

  • I. Stansfield
    • 1
  • Akhmaloka
    • 1
  • M. F. Tuite
    • 1
  1. 1.Research School of BiosciencesUniversity of KentCanterburyUK
  2. 2.Bandung Institute of TechnologyBandungIndonesia

Personalised recommendations