Isolation and functional characterization of a temperature-sensitive mutant of the yeast Saccharomyces cerevisiae in translation initiation factor eIF5: an eIF5-dependent cell-free translation system.
Eukaryotic translation initiation factor 5 (eIF5) interacts with the 40S ribosomal initiation complex (40S.eIF3.AUG.Met-tRNA(f).eIF2.GTP) to promote the hydrolysis of bound GTP. In Saccharomyces cerevisiae, eIF5, a protein of 45346Da, is encoded by a single-copy essential gene, TIF5. In this paper, we have isolated a temperature-sensitive S. cerevisiae strain, TMY5-1, by replacing the wild-type chromosomal copy of TIF5 with one mutagenized in vitro. The mutant yeast cells rapidly cease protein synthesis when grown under non-permissive conditions, lose polyribosomes and accumulate free 80S ribosomes. Further characterization of mutant eIF5 showed that the mutant protein, expressed in Escherichia coli, is defective both in its interaction with eIF2 as well as in mediating the hydrolysis of GTP bound to the 40S initiation complex and consequently in the formation of the 80S initiation complex. Additionally, the availability of a yeast strain containing temperature-sensitive mutation in the eIF5 gene allowed us to construct a cell-free translation system that was dependent on exogenously added eIF5 for translation of mRNAs in vitro.
Maiti
T
T
Department of Developmental and Molecular Biology, Albert Einstein College of Medicine of Yeshiva University, Jack and Pearl Resnick Campus, Bronx, NY 10461, USA.
Das
S
S
Maitra
U
U
D002474:Cell-Free System; D004926:Escherichia coli / Q000235:genetics; D015852:Eukaryotic Initiation Factor-2 / Q000378:metabolism; D040061:Eukaryotic Initiation Factor-5; D005800:Genes, Fungal / Q000235:genetics; D005982:Glutathione Transferase / Q000235:genetics; D006160:Guanosine Triphosphate / Q000378:metabolism; D006868:Hydrolysis; D016296:Mutagenesis; D009154:Mutation; D010448:Peptide Initiation Factors / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D011485:Protein Binding; D014176:Protein Biosynthesis; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012269:Ribosomal Proteins / Q000378:metabolism; D012270:Ribosomes / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D013696:Temperature
D015852:Eukaryotic Initiation Factor-2; D040061:Eukaryotic Initiation Factor-5; D010448:Peptide Initiation Factors; D011993:Recombinant Fusion Proteins; D012269:Ribosomal Proteins; D006160:Guanosine Triphosphate; D005982:Glutathione Transferase