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. 1988 Dec;120(4):923–934. doi: 10.1093/genetics/120.4.923

Mutations in Elongation Factor Ef-1α Affect the Frequency of Frameshifting and Amino Acid Misincorporation in Saccharomyces Cerevisiae

M G Sandbaken 1, M R Culbertson 1
PMCID: PMC1203584  PMID: 3066688

Abstract

A mutational analysis of the eukaryotic elongation factor EF-1α indicates that this protein functions to limit the frequency of errors during genetic code translation. We found that both amino acid misincorporation and reading frame errors are controlled by EF-1α. In order to examine the function of this protein, the TEF2 gene, which encodes EF-1α in Saccharomyces cerevisiae, was mutagenized in vitro with hydroxylamine. Sixteen independent TEF2 alleles were isolated by their ability to suppress frameshift mutations. DNA sequence analysis identified eight different sites in the EF-1α protein that elevate the frequency of mistranslation when mutated. These sites are located in two different regions of the protein. Amino acid substitutions located in or near the GTP-binding and hydrolysis domain of the protein cause suppression of frameshift and nonsense mutations. These mutations may effect mistranslation by altering the binding or hydrolysis of GTP. Amino acid substitutions located adjacent to a putative aminoacyl-tRNA binding region also suppress frameshift and nonsense mutations. These mutations may alter the binding of aminoacyl-tRNA by EF-1α. The identification of frameshift and nonsense suppressor mutations in EF-1α indicates a role for this protein in limiting amino acid misincorporation and reading frame errors. We suggest that these types of errors are controlled by a common mechanism or closely related mechanisms.

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Selected References

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  1. Ahn B. Y., Livingston D. M. Mitotic gene conversion lengths, coconversion patterns, and the incidence of reciprocal recombination in a Saccharomyces cerevisiae plasmid system. Mol Cell Biol. 1986 Nov;6(11):3685–3693. doi: 10.1128/mcb.6.11.3685. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Atkins J. F., Elseviers D., Gorini L. Low activity of -galactosidase in frameshift mutants of Escherichia coli. Proc Natl Acad Sci U S A. 1972 May;69(5):1192–1195. doi: 10.1073/pnas.69.5.1192. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Atkins J. F., Ryce S. UGA and non-triplet suppressor reading of the genetic code. Nature. 1974 Jun 7;249(457):527–530. doi: 10.1038/249527a0. [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. Botstein D., Falco S. C., Stewart S. E., Brennan M., Scherer S., Stinchcomb D. T., Struhl K., Davis R. W. Sterile host yeasts (SHY): a eukaryotic system of biological containment for recombinant DNA experiments. Gene. 1979 Dec;8(1):17–24. doi: 10.1016/0378-1119(79)90004-0. [DOI] [PubMed] [Google Scholar]
  6. Broach J. R., Strathern J. N., Hicks J. B. Transformation in yeast: development of a hybrid cloning vector and isolation of the CAN1 gene. Gene. 1979 Dec;8(1):121–133. doi: 10.1016/0378-1119(79)90012-x. [DOI] [PubMed] [Google Scholar]
  7. Bruce A. G., Atkins J. F., Gesteland R. F. tRNA anticodon replacement experiments show that ribosomal frameshifting can be caused by doublet decoding. Proc Natl Acad Sci U S A. 1986 Jul;83(14):5062–5066. doi: 10.1073/pnas.83.14.5062. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Busby S., Irani M., Crombrugghe B. Isolation of mutant promoters in the Escherichia coli galactose operon using local mutagenesis on cloned DNA fragments. J Mol Biol. 1982 Jan 15;154(2):197–209. doi: 10.1016/0022-2836(82)90060-2. [DOI] [PubMed] [Google Scholar]
  9. Chou P. Y., Fasman G. D. Prediction of the secondary structure of proteins from their amino acid sequence. Adv Enzymol Relat Areas Mol Biol. 1978;47:45–148. doi: 10.1002/9780470122921.ch2. [DOI] [PubMed] [Google Scholar]
  10. Clanton D. J., Hattori S., Shih T. Y. Mutations of the ras gene product p21 that abolish guanine nucleotide binding. Proc Natl Acad Sci U S A. 1986 Jul;83(14):5076–5080. doi: 10.1073/pnas.83.14.5076. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Cottrelle P., Thiele D., Price V. L., Memet S., Micouin J. Y., Marck C., Buhler J. M., Sentenac A., Fromageot P. Cloning, nucleotide sequence, and expression of one of two genes coding for yeast elongation factor 1 alpha. J Biol Chem. 1985 Mar 10;260(5):3090–3096. [PubMed] [Google Scholar]
  12. Dasmahapatra B., Chakraburtty K. Protein synthesis in yeast. I. Purification and properties of elongation factor 3 from Saccharomyces cerevisiae. J Biol Chem. 1981 Oct 10;256(19):9999–10004. [PubMed] [Google Scholar]
  13. Devereux J., Haeberli P., Smithies O. A comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Res. 1984 Jan 11;12(1 Pt 1):387–395. doi: 10.1093/nar/12.1part1.387. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Donahue T. F., Farabaugh P. J., Fink G. R. Suppressible four-base glycine and proline codons in yeast. Science. 1981 Apr 24;212(4493):455–457. doi: 10.1126/science.7010605. [DOI] [PubMed] [Google Scholar]
  15. Gaber R. F., Culbertson M. R. Frameshift suppression in Saccharomyces cerevisiae. IV. New suppressors among spontaneous co-revertants of the Group II his4-206 and leu 2-3 frameshift mutations. Genetics. 1982 Jul-Aug;101(3-4):345–367. doi: 10.1093/genetics/101.3-4.345. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Garnier J., Osguthorpe D. J., Robson B. Analysis of the accuracy and implications of simple methods for predicting the secondary structure of globular proteins. J Mol Biol. 1978 Mar 25;120(1):97–120. doi: 10.1016/0022-2836(78)90297-8. [DOI] [PubMed] [Google Scholar]
  17. Holmes D. S., Quigley M. A rapid boiling method for the preparation of bacterial plasmids. Anal Biochem. 1981 Jun;114(1):193–197. doi: 10.1016/0003-2697(81)90473-5. [DOI] [PubMed] [Google Scholar]
  18. Hughes D., Atkins J. F., Thompson S. Mutants of elongation factor Tu promote ribosomal frameshifting and nonsense readthrough. EMBO J. 1987 Dec 20;6(13):4235–4239. doi: 10.1002/j.1460-2075.1987.tb02772.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Hughes D. Mutant forms of tufA and tufB independently suppress nonsense mutations. J Mol Biol. 1987 Oct 20;197(4):611–615. doi: 10.1016/0022-2836(87)90467-0. [DOI] [PubMed] [Google Scholar]
  20. Hwang Y. W., Miller D. L. A mutation that alters the nucleotide specificity of elongation factor Tu, a GTP regulatory protein. J Biol Chem. 1987 Sep 25;262(27):13081–13085. [PubMed] [Google Scholar]
  21. 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]
  22. Jurnak F. Structure of the GDP domain of EF-Tu and location of the amino acids homologous to ras oncogene proteins. Science. 1985 Oct 4;230(4721):32–36. doi: 10.1126/science.3898365. [DOI] [PubMed] [Google Scholar]
  23. Lin J. P., Aker M., Sitney K. C., Mortimer R. K. First position wobble in codon-anticodon pairing: amber suppression by a yeast glutamine tRNA. Gene. 1986;49(3):383–388. doi: 10.1016/0378-1119(86)90375-6. [DOI] [PubMed] [Google Scholar]
  24. Mortimer R. K., Schild D. Genetic map of Saccharomyces cerevisiae, edition 9. Microbiol Rev. 1985 Sep;49(3):181–213. doi: 10.1128/mr.49.3.181-213.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Nasmyth K. A., Reed S. I. Isolation of genes by complementation in yeast: molecular cloning of a cell-cycle gene. Proc Natl Acad Sci U S A. 1980 Apr;77(4):2119–2123. doi: 10.1073/pnas.77.4.2119. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Orr-Weaver T. L., Szostak J. W., Rothstein R. J. Genetic applications of yeast transformation with linear and gapped plasmids. Methods Enzymol. 1983;101:228–245. doi: 10.1016/0076-6879(83)01017-4. [DOI] [PubMed] [Google Scholar]
  27. Orr-Weaver T. L., Szostak J. W., Rothstein R. J. Yeast transformation: a model system for the study of recombination. Proc Natl Acad Sci U S A. 1981 Oct;78(10):6354–6358. doi: 10.1073/pnas.78.10.6354. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. 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]
  29. Pure G. A., Robinson G. W., Naumovski L., Friedberg E. C. Partial suppression of an ochre mutation in Saccharomyces cerevisiae by multicopy plasmids containing a normal yeast tRNAGln gene. J Mol Biol. 1985 May 5;183(1):31–42. doi: 10.1016/0022-2836(85)90278-5. [DOI] [PubMed] [Google Scholar]
  30. Qin S. L., Moldave K., McLaughlin C. S. Isolation of the yeast gene encoding elongation factor 3 for protein synthesis. J Biol Chem. 1987 Jun 5;262(16):7802–7807. [PubMed] [Google Scholar]
  31. Rose M. D., Fink G. R. KAR1, a gene required for function of both intranuclear and extranuclear microtubules in yeast. Cell. 1987 Mar 27;48(6):1047–1060. doi: 10.1016/0092-8674(87)90712-4. [DOI] [PubMed] [Google Scholar]
  32. Rothstein R. J. One-step gene disruption in yeast. Methods Enzymol. 1983;101:202–211. doi: 10.1016/0076-6879(83)01015-0. [DOI] [PubMed] [Google Scholar]
  33. Schirmaier F., Philippsen P. Identification of two genes coding for the translation elongation factor EF-1 alpha of S. cerevisiae. EMBO J. 1984 Dec 20;3(13):3311–3315. doi: 10.1002/j.1460-2075.1984.tb02295.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Singh A., Ursic D., Davies J. Phenotypic suppression and misreading Saccharomyces cerevisiae. Nature. 1979 Jan 11;277(5692):146–148. doi: 10.1038/277146a0. [DOI] [PubMed] [Google Scholar]
  35. Staden R. An interactive graphics program for comparing and aligning nucleic acid and amino acid sequences. Nucleic Acids Res. 1982 May 11;10(9):2951–2961. doi: 10.1093/nar/10.9.2951. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Stinchcomb D. T., Mann C., Davis R. W. Centromeric DNA from Saccharomyces cerevisiae. J Mol Biol. 1982 Jun 25;158(2):157–190. doi: 10.1016/0022-2836(82)90427-2. [DOI] [PubMed] [Google Scholar]
  37. Tapio S., Kurland C. G. Mutant EF-Tu increases missense error in vitro. Mol Gen Genet. 1986 Oct;205(1):186–188. doi: 10.1007/BF02428051. [DOI] [PubMed] [Google Scholar]
  38. 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]
  39. Uritani M., Miyazaki M. Role of yeast peptide elongation factor 3 (EF-3) at the AA-tRNA binding step. J Biochem. 1988 Jul;104(1):118–126. doi: 10.1093/oxfordjournals.jbchem.a122405. [DOI] [PubMed] [Google Scholar]
  40. Van Noort J. M., Kraal B., Bosch L. A second tRNA binding site on elongation factor Tu is induced while the factor is bound to the ribosome. Proc Natl Acad Sci U S A. 1985 May;82(10):3212–3216. doi: 10.1073/pnas.82.10.3212. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. 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]
  42. 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]
  43. Weiss R. B., Gallant J. A. Frameshift suppression in aminoacyl-tRNA limited cells. Genetics. 1986 Apr;112(4):727–739. doi: 10.1093/genetics/112.4.727. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Weiss W. A., Edelman I., Culbertson M. R., Friedberg E. C. Physiological levels of normal tRNA(CAGGln) can effect partial suppression of amber mutations in the yeast Saccharomyces cerevisiae. Proc Natl Acad Sci U S A. 1987 Nov;84(22):8031–8034. doi: 10.1073/pnas.84.22.8031. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Weiss W. A., Friedberg E. C. Normal yeast tRNA(CAGGln) can suppress amber codons and is encoded by an essential gene. J Mol Biol. 1986 Dec 20;192(4):725–735. doi: 10.1016/0022-2836(86)90024-0. [DOI] [PubMed] [Google Scholar]
  46. Wolf H., Modrow S., Motz M., Jameson B. A., Hermann G., Förtsch B. An integrated family of amino acid sequence analysis programs. Comput Appl Biosci. 1988 Mar;4(1):187–191. doi: 10.1093/bioinformatics/4.1.187. [DOI] [PubMed] [Google Scholar]

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