TY - JOUR
T1 - Isolation and functional characterization of a temperature-sensitive mutant of the yeast Saccharomyces cerevisiae in translation initiation factor eIF5
T2 - An eIF5-dependent cell-free translation system
AU - Maiti, Tapan
AU - Das, Supratik
AU - Maitra, Umadas
N1 - Funding Information:
We are indebted to Dr Gregory Prelich of this institution for many helpful discussions on the construction of temperature-sensitive yeast mutants and to Dr Philip Hieter, Johns Hopkins University School of Medicine, Baltimore, MD, for providing us with the yeast plasmids pRS315 and pRS316. This research was supported by grant GM15399 from the National Institutes of Health, USA and Cancer Core Support grant P30CA13330 from the National Cancer Institute, USA.
PY - 2000/2/22
Y1 - 2000/2/22
N2 - 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 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 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. (C) 2000 Elsevier Science B.V.
AB - 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 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 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. (C) 2000 Elsevier Science B.V.
KW - Protein synthesis
KW - Ribsosome
KW - Translation initiation factors
KW - Yeast
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U2 - 10.1016/S0378-1119(99)00570-3
DO - 10.1016/S0378-1119(99)00570-3
M3 - Article
C2 - 10689193
AN - SCOPUS:0033975909
SN - 0378-1119
VL - 244
SP - 109
EP - 118
JO - Gene
JF - Gene
IS - 1-2
ER -