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. 1989 Oct;9(10):4467–4472. doi: 10.1128/mcb.9.10.4467

Translation in Saccharomyces cerevisiae: initiation factor 4E-dependent cell-free system.

M Altmann 1, N Sonenberg 1, H Trachsel 1
PMCID: PMC362530  PMID: 2685552

Abstract

The gene encoding translation initiation factor 4E (eIF-4E) from Saccharomyces cerevisiae was randomly mutagenized in vitro. The mutagenized gene was reintroduced on a plasmid into S. cerevisiae cells having their only wild-type eIF-4E gene on a plasmid under the control of the regulatable GAL1 promoter. Transcription from the GAL1 promoter (and consequently the production of wild-type eIF-4E) was then shut off by plating these cells on glucose-containing medium. Under these conditions, the phenotype conferred upon the cells by the mutated eIF-4E gene became apparent. Temperature-sensitive S. cerevisiae strains were identified by replica plating. The properties of one strain, 4-2, were further analyzed. Strain 4-2 has two point mutations in the eIF-4E gene. Upon incubation at 37 degrees C, incorporation of [35S]methionine was reduced to 15% of the wild-type level. Cell-free translation systems derived from strain 4-2 were dependent on exogenous eIF-4E for efficient translation of certain mRNAs, and this dependence was enhanced by preincubation of the extract at 37 degrees C. Not all mRNAs tested required exogenous eIF-4E for translation.

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Selected References

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

  1. Altmann M., Edery I., Sonenberg N., Trachsel H. Purification and characterization of protein synthesis initiation factor eIF-4E from the yeast Saccharomyces cerevisiae. Biochemistry. 1985 Oct 22;24(22):6085–6089. doi: 10.1021/bi00343a009. [DOI] [PubMed] [Google Scholar]
  2. Altmann M., Edery I., Trachsel H., Sonenberg N. Site-directed mutagenesis of the tryptophan residues in yeast eukaryotic initiation factor 4E. Effects on cap binding activity. J Biol Chem. 1988 Nov 25;263(33):17229–17232. [PubMed] [Google Scholar]
  3. Altmann M., Handschin C., Trachsel H. mRNA cap-binding protein: cloning of the gene encoding protein synthesis initiation factor eIF-4E from Saccharomyces cerevisiae. Mol Cell Biol. 1987 Mar;7(3):998–1003. doi: 10.1128/mcb.7.3.998. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Anderson C. W., Baum P. R., Gesteland R. F. Processing of adenovirus 2-induced proteins. J Virol. 1973 Aug;12(2):241–252. doi: 10.1128/jvi.12.2.241-252.1973. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Buckley B., Ehrenfeld E. The cap-binding protein complex in uninfected and poliovirus-infected HeLa cells. J Biol Chem. 1987 Oct 5;262(28):13599–13606. [PubMed] [Google Scholar]
  6. Chamberlain J. P. Fluorographic detection of radioactivity in polyacrylamide gels with the water-soluble fluor, sodium salicylate. Anal Biochem. 1979 Sep 15;98(1):132–135. doi: 10.1016/0003-2697(79)90716-4. [DOI] [PubMed] [Google Scholar]
  7. 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]
  8. Edery I., Altmann M., Sonenberg N. High-level synthesis in Escherichia coli of functional cap-binding eukaryotic initiation factor eIF-4E and affinity purification using a simplified cap-analog resin. Gene. 1988 Dec 30;74(2):517–525. doi: 10.1016/0378-1119(88)90184-9. [DOI] [PubMed] [Google Scholar]
  9. Edery I., Hümbelin M., Darveau A., Lee K. A., Milburn S., Hershey J. W., Trachsel H., Sonenberg N. Involvement of eukaryotic initiation factor 4A in the cap recognition process. J Biol Chem. 1983 Sep 25;258(18):11398–11403. [PubMed] [Google Scholar]
  10. Edery I., Lee K. A., Sonenberg N. Functional characterization of eukaryotic mRNA cap binding protein complex: effects on translation of capped and naturally uncapped RNAs. Biochemistry. 1984 May 22;23(11):2456–2462. doi: 10.1021/bi00306a021. [DOI] [PubMed] [Google Scholar]
  11. Gasior E., Herrera F., Sadnik I., McLaughlin C. S., Moldave K. The preparation and characterization of a cell-free system from Saccharomyces cerevisiae that translates natural messenger ribonucleic acid. J Biol Chem. 1979 May 25;254(10):3965–3969. [PubMed] [Google Scholar]
  12. Goyer C., Altmann M., Trachsel H., Sonenberg N. Identification and characterization of cap-binding proteins from yeast. J Biol Chem. 1989 May 5;264(13):7603–7610. [PubMed] [Google Scholar]
  13. Kozak M. Comparison of initiation of protein synthesis in procaryotes, eucaryotes, and organelles. Microbiol Rev. 1983 Mar;47(1):1–45. doi: 10.1128/mr.47.1.1-45.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Kozak M. Effects of intercistronic length on the efficiency of reinitiation by eucaryotic ribosomes. Mol Cell Biol. 1987 Oct;7(10):3438–3445. doi: 10.1128/mcb.7.10.3438. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Linder P., Slonimski P. P. Sequence of the genes TIF1 and TIF2 from Saccharomyces cerevisiae coding for a translation initiation factor. Nucleic Acids Res. 1988 Nov 11;16(21):10359–10359. doi: 10.1093/nar/16.21.10359. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. 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]
  17. Mandel T., Trachsel H. Yeast, Saccharomyces cerevisiae, cell-free translation: the inhibition of translation by high temperature is reversible. Biochim Biophys Acta. 1989 Jan 23;1007(1):80–83. doi: 10.1016/0167-4781(89)90133-4. [DOI] [PubMed] [Google Scholar]
  18. Pain V. M. Initiation of protein synthesis in mammalian cells. Biochem J. 1986 May 1;235(3):625–637. doi: 10.1042/bj2350625. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. 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]
  20. 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]
  21. Sleat D. E., Gallie D. R., Jefferson R. A., Bevan M. W., Turner P. C., Wilson T. M. Characterisation of the 5'-leader sequence of tobacco mosaic virus RNA as a general enhancer of translation in vitro. Gene. 1987;60(2-3):217–225. doi: 10.1016/0378-1119(87)90230-7. [DOI] [PubMed] [Google Scholar]
  22. Sonenberg N., Guertin D., Lee K. A. Capped mRNAs with reduced secondary structure can function in extracts from poliovirus-infected cells. Mol Cell Biol. 1982 Dec;2(12):1633–1638. doi: 10.1128/mcb.2.12.1633. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Sonenberg N. Regulation of translation by poliovirus. Adv Virus Res. 1987;33:175–204. doi: 10.1016/s0065-3527(08)60318-8. [DOI] [PubMed] [Google Scholar]
  24. Towbin H., Staehelin T., Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4350–4354. doi: 10.1073/pnas.76.9.4350. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Yaffe M. P., Schatz G. Two nuclear mutations that block mitochondrial protein import in yeast. Proc Natl Acad Sci U S A. 1984 Aug;81(15):4819–4823. doi: 10.1073/pnas.81.15.4819. [DOI] [PMC free article] [PubMed] [Google Scholar]

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