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. 1992 Sep;132(1):97–112. doi: 10.1093/genetics/132.1.97

Cis- and Trans-Acting Suppressors of a Translation Initiation Defect at the Cyc1 Locus of Saccharomyces Cerevisiae

I Pinto 1, J G Na 1, F Sherman 1, M Hampsey 1
PMCID: PMC1205134  PMID: 1327957

Abstract

The cyc1-362 mutant of Saccharomyces cerevisiae is deficient in iso-1-cytochrome c as a consequence of an aberrant ATG codon that initiates a short open reading frame (uORF) in the cyc1 transcribed leader region. We have isolated and characterized functional revertants of cyc1-362 in an effort to define cis- and trans-acting factors that can suppress the effect of the uORF. Genetic and DNA sequence analyses have defined three classes of revertants: (i) those that acquired point mutations in the upstream ATG (uATG), restoring iso-1-cytochrome c to its normal level; (ii) substitution of the normal A residue at position -1 relative to the uATG by either C or T, enhancing iso-1-cytochrome c production from &2% to 6% (C) or 10% (T) of normal, indicating that the nucleotide immediately preceding the initiator codon can affect the efficiency of AUG start codon recognition and that purines are preferred over pyrimidines at this site; and (iii) extragenic suppressors that enhance iso-1-cytochrome c expression to 10-40% of normal while retaining the uATG. These suppressors are represented by five different genes, designated sua1-sua4 and sua6. In contrast to the previously described sua7 and sua8 suppressors, they do not compensate for the uATG by affecting cyc1 transcription start site selection. Potential suppressor mechanisms are discussed.

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Selected References

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  1. Baim S. B., Sherman F. mRNA structures influencing translation in the yeast Saccharomyces cerevisiae. Mol Cell Biol. 1988 Apr;8(4):1591–1601. doi: 10.1128/mcb.8.4.1591. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Barry K., Stiles J. I., Pietras D. F., Melnick L., Sherman F. Physical analysis of the COR region: a cluster of six genes in Saccharomyces cerevisiae. Mol Cell Biol. 1987 Feb;7(2):632–638. doi: 10.1128/mcb.7.2.632. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Berroteran R. W., Hampsey M. Genetic analysis of yeast iso-1-cytochrome c structural requirements: suppression of Gly6 replacements by an Asn52----Ile replacement. Arch Biochem Biophys. 1991 Jul;288(1):261–269. doi: 10.1016/0003-9861(91)90193-m. [DOI] [PubMed] [Google Scholar]
  4. Birnboim H. C., Doly J. A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res. 1979 Nov 24;7(6):1513–1523. doi: 10.1093/nar/7.6.1513. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Castilho-Valavicius B., Yoon H., Donahue T. F. Genetic characterization of the Saccharomyces cerevisiae translational initiation suppressors sui1, sui2 and SUI3 and their effects on HIS4 expression. Genetics. 1990 Mar;124(3):483–495. doi: 10.1093/genetics/124.3.483. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Cavener D. R., Ray S. C. Eukaryotic start and stop translation sites. Nucleic Acids Res. 1991 Jun 25;19(12):3185–3192. doi: 10.1093/nar/19.12.3185. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Cigan A. M., Feng L., Donahue T. F. tRNAi(met) functions in directing the scanning ribosome to the start site of translation. Science. 1988 Oct 7;242(4875):93–97. doi: 10.1126/science.3051379. [DOI] [PubMed] [Google Scholar]
  8. Cigan A. M., Pabich E. K., Feng L., Donahue T. F. Yeast translation initiation suppressor sui2 encodes the alpha subunit of eukaryotic initiation factor 2 and shares sequence identity with the human alpha subunit. Proc Natl Acad Sci U S A. 1989 Apr;86(8):2784–2788. doi: 10.1073/pnas.86.8.2784. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Clements J. M., Laz T. M., Sherman F. Efficiency of translation initiation by non-AUG codons in Saccharomyces cerevisiae. Mol Cell Biol. 1988 Oct;8(10):4533–4536. doi: 10.1128/mcb.8.10.4533. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Curran J., Kolakofsky D. Scanning independent ribosomal initiation of the Sendai virus Y proteins in vitro and in vivo. EMBO J. 1989 Feb;8(2):521–526. doi: 10.1002/j.1460-2075.1989.tb03406.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Donahue T. F., Cigan A. M., Pabich E. K., Valavicius B. C. Mutations at a Zn(II) finger motif in the yeast eIF-2 beta gene alter ribosomal start-site selection during the scanning process. Cell. 1988 Aug 26;54(5):621–632. doi: 10.1016/s0092-8674(88)80006-0. [DOI] [PubMed] [Google Scholar]
  12. Emr S. D., Vassarotti A., Garrett J., Geller B. L., Takeda M., Douglas M. G. The amino terminus of the yeast F1-ATPase beta-subunit precursor functions as a mitochondrial import signal. J Cell Biol. 1986 Feb;102(2):523–533. doi: 10.1083/jcb.102.2.523. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Errede B., Cardillo T. S., Wever G., Sherman F., Stiles J. I., Friedman L. R., Sherman F. Studies on transposable elements in yeast. I. ROAM mutations causing increased expression of yeast genes: their activation by signals directed toward conjugation functions and their formation by insertion of Ty1 repetitive elements. II. deletions, duplications, and transpositions of the COR segment that encompasses the structural gene of yeast iso-1-cytochrome c. Cold Spring Harb Symp Quant Biol. 1981;45(Pt 2):593–607. [PubMed] [Google Scholar]
  14. Guarente L. Yeast promoters and lacZ fusions designed to study expression of cloned genes in yeast. Methods Enzymol. 1983;101:181–191. doi: 10.1016/0076-6879(83)01013-7. [DOI] [PubMed] [Google Scholar]
  15. Hampsey D. M., Das G., Sherman F. Amino acid replacements in yeast iso-1-cytochrome c. Comparison with the phylogenetic series and the tertiary structure of related cytochromes c. J Biol Chem. 1986 Mar 5;261(7):3259–3271. [PubMed] [Google Scholar]
  16. Hampsey M., Na J. G., Pinto I., Ware D. E., Berroteran R. W. Extragenic suppressors of a translation initiation defect in the cyc1 gene of Saccharomyces cerevisiae. Biochimie. 1991 Dec;73(12):1445–1455. doi: 10.1016/0300-9084(91)90177-3. [DOI] [PubMed] [Google Scholar]
  17. Holm C., Meeks-Wagner D. W., Fangman W. L., Botstein D. A rapid, efficient method for isolating DNA from yeast. Gene. 1986;42(2):169–173. doi: 10.1016/0378-1119(86)90293-3. [DOI] [PubMed] [Google Scholar]
  18. 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]
  19. Jang S. K., Kräusslich H. G., Nicklin M. J., Duke G. M., Palmenberg A. C., Wimmer E. A segment of the 5' nontranslated region of encephalomyocarditis virus RNA directs internal entry of ribosomes during in vitro translation. J Virol. 1988 Aug;62(8):2636–2643. doi: 10.1128/jvi.62.8.2636-2643.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Kozak M. An analysis of 5'-noncoding sequences from 699 vertebrate messenger RNAs. Nucleic Acids Res. 1987 Oct 26;15(20):8125–8148. doi: 10.1093/nar/15.20.8125. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Kozak M. Bifunctional messenger RNAs in eukaryotes. Cell. 1986 Nov 21;47(4):481–483. doi: 10.1016/0092-8674(86)90609-4. [DOI] [PubMed] [Google Scholar]
  22. Kozak M. Evaluation of the "scanning model" for initiation of protein synthesis in eucaryotes. Cell. 1980 Nov;22(1 Pt 1):7–8. doi: 10.1016/0092-8674(80)90148-8. [DOI] [PubMed] [Google Scholar]
  23. Kozak M. How do eucaryotic ribosomes select initiation regions in messenger RNA? Cell. 1978 Dec;15(4):1109–1123. doi: 10.1016/0092-8674(78)90039-9. [DOI] [PubMed] [Google Scholar]
  24. Kozak M. The scanning model for translation: an update. J Cell Biol. 1989 Feb;108(2):229–241. doi: 10.1083/jcb.108.2.229. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Kunkel T. A., Roberts J. D., Zakour R. A. Rapid and efficient site-specific mutagenesis without phenotypic selection. Methods Enzymol. 1987;154:367–382. doi: 10.1016/0076-6879(87)54085-x. [DOI] [PubMed] [Google Scholar]
  26. Lawrence C. W., Stewart J. W., Sherman F., Christensen R. Specificity and frequency of ultraviolet-induced reversion of an iso-1-cytochrome c ochre mutant in radiation-sensitive strains of yeast. J Mol Biol. 1974 May 5;85(1):137–162. doi: 10.1016/0022-2836(74)90134-x. [DOI] [PubMed] [Google Scholar]
  27. Leeds P., Peltz S. W., Jacobson A., Culbertson M. R. The product of the yeast UPF1 gene is required for rapid turnover of mRNAs containing a premature translational termination codon. Genes Dev. 1991 Dec;5(12A):2303–2314. doi: 10.1101/gad.5.12a.2303. [DOI] [PubMed] [Google Scholar]
  28. Li W. Z., Sherman F. Two types of TATA elements for the CYC1 gene of the yeast Saccharomyces cerevisiae. Mol Cell Biol. 1991 Feb;11(2):666–676. doi: 10.1128/mcb.11.2.666. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Liu C. C., Simonsen C. C., Levinson A. D. Initiation of translation at internal AUG codons in mammalian cells. Nature. 1984 May 3;309(5963):82–85. doi: 10.1038/309082a0. [DOI] [PubMed] [Google Scholar]
  30. Mandel M., Higa A. Calcium-dependent bacteriophage DNA infection. J Mol Biol. 1970 Oct 14;53(1):159–162. doi: 10.1016/0022-2836(70)90051-3. [DOI] [PubMed] [Google Scholar]
  31. McKnight G. L., Cardillo T. S., Sherman F. An extensive deletion causing overproduction of yeast iso-2-cytochrome c. Cell. 1981 Aug;25(2):409–419. doi: 10.1016/0092-8674(81)90059-3. [DOI] [PubMed] [Google Scholar]
  32. McNeil J. B., Smith M. Transcription initiation of the Saccharomyces cerevisiae iso-1-cytochrome c gene. Multiple, independent T-A-T-A sequences. J Mol Biol. 1986 Feb 5;187(3):363–378. doi: 10.1016/0022-2836(86)90439-0. [DOI] [PubMed] [Google Scholar]
  33. Melnick L., Sherman F. Nucleotide sequence of the COR region: a cluster of six genes in the yeast Saccharomyces cerevisiae. Gene. 1990 Mar 15;87(2):157–166. doi: 10.1016/0378-1119(90)90297-5. [DOI] [PubMed] [Google Scholar]
  34. Müller P. P., Trachsel H. Translation and regulation of translation in the yeast Saccharomyces cerevisiae. Eur J Biochem. 1990 Jul 31;191(2):257–261. doi: 10.1111/j.1432-1033.1990.tb19118.x. [DOI] [PubMed] [Google Scholar]
  35. Na J. G., Pinto I., Hampsey M. Isolation and characterization of SUA5, a novel gene required for normal growth in Saccharomyces cerevisiae. Genetics. 1992 Aug;131(4):791–801. doi: 10.1093/genetics/131.4.791. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Peabody D. S., Berg P. Termination-reinitiation occurs in the translation of mammalian cell mRNAs. Mol Cell Biol. 1986 Jul;6(7):2695–2703. doi: 10.1128/mcb.6.7.2695. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Pelletier J., Sonenberg N. Internal initiation of translation of eukaryotic mRNA directed by a sequence derived from poliovirus RNA. Nature. 1988 Jul 28;334(6180):320–325. doi: 10.1038/334320a0. [DOI] [PubMed] [Google Scholar]
  38. Pinto I., Ware D. E., Hampsey M. The yeast SUA7 gene encodes a homolog of human transcription factor TFIIB and is required for normal start site selection in vivo. Cell. 1992 Mar 6;68(5):977–988. doi: 10.1016/0092-8674(92)90040-j. [DOI] [PubMed] [Google Scholar]
  39. Rose M., Casadaban M. J., Botstein D. Yeast genes fused to beta-galactosidase in Escherichia coli can be expressed normally in yeast. Proc Natl Acad Sci U S A. 1981 Apr;78(4):2460–2464. doi: 10.1073/pnas.78.4.2460. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Rothstein R. J., Sherman F. Genes affecting the expression of cytochrome c in yeast: genetic mapping and genetic interactions. Genetics. 1980 Apr;94(4):871–889. doi: 10.1093/genetics/94.4.871. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. 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]
  42. Sherman F. Getting started with yeast. Methods Enzymol. 1991;194:3–21. doi: 10.1016/0076-6879(91)94004-v. [DOI] [PubMed] [Google Scholar]
  43. Sherman F., Hicks J. Micromanipulation and dissection of asci. Methods Enzymol. 1991;194:21–37. doi: 10.1016/0076-6879(91)94005-w. [DOI] [PubMed] [Google Scholar]
  44. Sherman F., Stewart J. W., Jackson M., Gilmore R. A., Parker J. H. Mutants of yeast defective in iso-1-cytochrome c. Genetics. 1974 Jun;77(2):255–284. doi: 10.1093/genetics/77.2.255. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Smith M., Leung D. W., Gillam S., Astell C. R., Montgomery D. L., Hall B. D. Sequence of the gene for iso-1-cytochrome c in Saccharomyces cerevisiae. Cell. 1979 Apr;16(4):753–761. doi: 10.1016/0092-8674(79)90091-6. [DOI] [PubMed] [Google Scholar]
  46. Stiles J. I., Szostak J. W., Young A. T., Wu R., Consaul S., Sherman F. DNA sequence of a mutation in the leader region of the yeast iso-1-cytochrome c mRNA. Cell. 1981 Jul;25(1):277–284. doi: 10.1016/0092-8674(81)90253-1. [DOI] [PubMed] [Google Scholar]
  47. Yoon H. J., Donahue T. F. The suil suppressor locus in Saccharomyces cerevisiae encodes a translation factor that functions during tRNA(iMet) recognition of the start codon. Mol Cell Biol. 1992 Jan;12(1):248–260. doi: 10.1128/mcb.12.1.248. [DOI] [PMC free article] [PubMed] [Google Scholar]

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