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
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
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DOI：
10.1016/s0378-1119(99)00570-3
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发表时间：
2000-02-22
期刊：
影响因子：
3.5
通讯作者：
Maitra, U
中科院分区：
文献类型：
--
作者：
Maiti, T;Das, S;Maitra, U
真核翻译起始因子5（eIF 5）与40 S核糖体起始复合物（40 S. eIF 3.AUG.Met-tRNA（f）. eIF 2. GTP）相互作用以促进结合的GTP的水解。在酿酒酵母（Saccharomyces cerevisiae）中，eIF 5是一种分子量为45 346 Da的蛋白质，由一个单拷贝必需基因TIF 5编码。本文分离了一株温度敏感的S.通过用体外诱变的TIF 5替换TIF 5的野生型染色体拷贝，将TIF 5转化至酿酒酵母菌株TMY 5 -1。当在非允许条件下生长时，突变酵母细胞迅速停止蛋白质合成，失去多聚核糖体并积累游离80 S核糖体。对突变体eIF 5的进一步表征表明，突变体蛋白。在大肠杆菌中表达，在与eIF 2的相互作用以及介导与40 S起始复合物结合的GTP的水解以及因此在80 S起始复合物的形成方面都有缺陷。另外。在eIF 5基因中含有温度敏感性突变的酵母菌株的可用性允许我们构建依赖于外源添加的eIF 5用于体外mRNA翻译的无细胞翻译系统。(C)2000由Elsevier Science B. V.出版，保留所有权利。
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 Published by Elsevier Science B.V. All rights reserved.