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 ri bosomal initiation complex (40S.eIF3.AUG.Met-tRNA(f).eIF2.GTP) to promote t he hydrolysis of bound GTP. In Saccharomyces cerevisiae, eIF5, a protein of 45 346 Da, 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 v itro. The mutant yeast cells rapidly cease protein synthesis when grown und er non-permissive conditions, lose polyribosomes and accumulate free 80S ri bosomes. Further characterization of mutant eIF5 showed that the mutant pro tein. expressed in Escherichia coli, is defective both in its interaction w ith eIF2 as well as in mediating the hydrolysis of GTP bound to the 40S ini tiation complex and consequently in the formation of the 80S initiation com plex. Additionally. the availability of a yeast strain containing temperatu re-sensitive mutation in the eIF5 gene allowed us to construct a cell-free translation system that was dependent on exogenously added eIF5 for transla tion of mRNAs in vitro. (C) 2000 Published by Elsevier Science B.V. All rig hts reserved.