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首页> 美国卫生研究院文献>Molecular and Cellular Biology >Suppressor analysis of temperature-sensitive mutations of the largest subunit of RNA polymerase I in Saccharomyces cerevisiae: a suppressor gene encodes the second-largest subunit of RNA polymerase I.
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Suppressor analysis of temperature-sensitive mutations of the largest subunit of RNA polymerase I in Saccharomyces cerevisiae: a suppressor gene encodes the second-largest subunit of RNA polymerase I.

机译：酿酒酵母（Saccharomyces cerevisiae）RNA聚合酶I最大亚基的温度敏感突变的抑制子分析：抑制基因编码RNA聚合酶I第二大亚基。

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The SRP3-1 mutation is an allele-specific suppressor of temperature-sensitive mutations in the largest subunit (A190) of RNA polymerase I from Saccharomyces cerevisiae. Two mutations known to be suppressed by SRP3-1 are in the putative zinc-binding domain of A190. We have cloned the SRP3 gene by using its suppressor activity and determined its complete nucleotide sequence. We conclude from the following evidence that the SRP3 gene encodes the second-largest subunit (A135) of RNA polymerase I. First, the deduced amino acid sequence of the gene product contains several regions with high homology to the corresponding regions of the second-largest subunits of RNA polymerases of various origins, including those of RNA polymerase II and III from S. cerevisiae. Second, the deduced amino acid sequence contains known amino acid sequences of two tryptic peptides from the A135 subunit of RNA polymerase I purified from S. cerevisiae. Finally, a strain was constructed in which transcription of the SRP3 gene was controlled by the inducible GAL7 promoter. When this strain, which can grow on galactose but not on glucose, was shifted from galactose medium to glucose medium, a large decrease in the cellular concentration of A135 was observed by Western blot analysis. We have also identified the specific amino acid alteration responsible for suppression by SRP3-1 and found that it is located within the putative zinc-binding domain conserved among the second-largest subunits of eucaryotic RNA polymerases. From these results, it is suggested that this putative zinc-binding domain is in physical proximity to and interacts with the putative zinc-binding domain of the A190 subunit.
机译：SRP3-1突变是酿酒酵母（Saccharomyces cerevisiae）RNA聚合酶I的最大亚基（A190）中温度敏感突变的等位基因特异性抑制剂。已知被SRP3-1抑制的两个突变位于A190的假定锌结合域中。我们已经利用其抑制活性克隆了SRP3基因，并确定了其完整的核苷酸序列。我们从以下证据得出结论，即SRP3基因编码RNA聚合酶I的第二大亚基（A135）。首先，推导的基因产物氨基酸序列包含与第二大亚基的相应区域具有高度同源性的几个区域各种来源的RNA聚合酶的亚基，包括酿酒酵母（S. cerevisiae）的RNA聚合酶II和III的亚基。其次，推导的氨基酸序列含有来自酿酒酵母RNA纯化的RNA聚合酶I的A135亚基的两个胰蛋白酶肽的已知氨基酸序列。最后，构建了一种菌株，其中SRP3基因的转录受诱导型GAL7启动子控制。当可以在半乳糖上生长但不能在葡萄糖上生长的该菌株从半乳糖培养基转移到葡萄糖培养基时，通过蛋白质印迹分析观察到A135的细胞浓度大大降低。我们还确定了由SRP3-1抑制的特定氨基酸改变，并发现它位于真核RNA聚合酶第二大亚基之间保守的推定锌结合域内。从这些结果表明，该推定的锌结合域与A190亚基的推定的锌结合域在物理上接近并相互作用。

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