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首页> 外文期刊>Molecular and Cellular Biology >Translation in Saccharomyces cerevisiae: initiation factor 4E-dependent cell-free system.
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Translation in Saccharomyces cerevisiae: initiation factor 4E-dependent cell-free system.

机译：酿酒酵母中的翻译：起始因子4E依赖的无细胞系统。

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摘要

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.
机译：来自啤酒酵母的翻译起始因子4E（eIF-4E）的基因在体外被随机诱变。将诱变的基因在质粒上重新引入到酿酒酵母细胞中，所述啤酒酵母细胞在可调节的GAL1启动子的控制下在质粒上具有唯一的野生型eIF-4E基因。然后，通过将这些细胞铺板在含葡萄糖的培养基上，关闭GAL1启动子的转录（进而产生野生型eIF-4E）。在这些条件下，突变的eIF-4E基因赋予细胞的表型变得很明显。通过复制平板鉴定温度敏感的酿酒酵母菌株。进一步分析了一种4-2菌株的特性。菌株4-2在eIF-4E基因中具有两个点突变。在37℃下温育后，[35S]蛋氨酸的掺入减少到野生型水平的15％。源自菌株4-2的无细胞翻译系统依赖于外源eIF-4E进行某些mRNA的有效翻译，并且通过在37摄氏度下预孵育提取物来增强这种依赖性。并非所有测试的mRNA都需要外源eIF-4E用于翻译。

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