Dever T E
Laboratory of Eukaryotic Gene Regulation, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892, USA.
Methods. 1997 Apr;11(4):403-17. doi: 10.1006/meth.1996.0437.
Molecular genetic analyses in yeast are a powerful method to study gene regulation. Conservation of the mechanism and regulation of protein synthesis between yeast and mammalian cells makes yeast a good model system for the analysis of translation. One of the most common mechanisms of translational regulation in mammalian cells is the phosphorylation of serine-51 on the alpha subunit of the translation initiation factor elF2, which causes an inhibition of general translation. In contrast, in the yeast Saccharomyces cerevisiae phosphorylation of elF2 alpha on serine-51 by the GCN2 protein kinase mediates the translational induction of GCN4 expression. The unique structure of the GCN4 mRNA makes GCN4 expression especially sensitive to elF2 alpha phosphorylation, and the simple microbiological tests developed in yeast to analyze GCN4 expression serve as good reporters of elF2 alpha phosphorylation. It is relatively simple to express heterologous proteins in yeast, and it has been shown that the mammalian elF2 alpha kinases will functionally substitute for GCN2. Structure-function analyses of translation factors or translational regulators can also be performed by assaying for effects on general and GCN4-specific translation. Three tests can be used to study elF2 alpha phosphorylation and/or translational activity in yeast. First, general translation can be monitored by simple growth tests, while GCN4 expression can be analyzed using sensitive replicaplating tests. Second, GCN4 translation can be quantitated by measuring expression from GCN4-lacZ reporter constructs. Finally, isoelectric focusing gels can be used to directly monitor in vivo phosphorylation of elF2 alpha in yeast.
酵母中的分子遗传学分析是研究基因调控的一种强大方法。酵母和哺乳动物细胞之间蛋白质合成机制及调控的保守性，使酵母成为分析翻译的良好模型系统。哺乳动物细胞中翻译调控最常见的机制之一是翻译起始因子elF2的α亚基上丝氨酸-51的磷酸化，这会导致整体翻译受到抑制。相比之下，在酿酒酵母中，GCN2蛋白激酶使elF2α的丝氨酸-51磷酸化，介导了GCN4表达的翻译诱导。GCN4 mRNA的独特结构使GCN4表达对elF2α磷酸化特别敏感，并且在酵母中开发的用于分析GCN4表达的简单微生物学测试可作为elF2α磷酸化的良好报告指标。在酵母中表达异源蛋白相对简单，并且已经表明哺乳动物的elF2α激酶在功能上可替代GCN2。翻译因子或翻译调节因子的结构-功能分析也可以通过检测对整体翻译和GCN4特异性翻译的影响来进行。可以使用三种测试来研究酵母中的elF2α磷酸化和/或翻译活性。首先，可以通过简单的生长测试监测整体翻译，而使用灵敏的复制平板测试分析GCN4表达。其次，可以通过测量GCN4-lacZ报告构建体的表达来定量GCN4翻译。最后，可以使用等电聚焦凝胶直接监测酵母中elF2α的体内磷酸化。
