Translation in Saccharomyces cerevisiae: initiation factor 4E-dependent cell-free system.
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.
Altmann
M
M
Institut für Biochemie und Molekularbiologie, Universität Bern, Switzerland.
Sonenberg
N
N
Trachsel
H
H
D000595:Amino Acid Sequence; D001483:Base Sequence; D039561:Eukaryotic Initiation Factor-4E; D008969:Molecular Sequence Data; D009154:Mutation; D010448:Peptide Initiation Factors / Q000502:physiology*; D014176:Protein Biosynthesis / Q000502:physiology*; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D013696:Temperature
D039561:Eukaryotic Initiation Factor-4E; D010448:Peptide Initiation Factors