Termination-altering mutations in the second-largest subunit of yeast RNA polymerase III.
Termination-altering mutations in the second-largest subunit of yeast RNA polymerase III.
复制标题
酵母 RNA 聚合酶 III 第二大亚基的终止改变突变。
DOI：
10.1128/mcb.15.3.1467
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发表时间：
1995
影响因子：
5.3
通讯作者：
Hall,BD
中科院分区：
文献类型：
--
作者：
Shaaban,SA;Krupp,BM;Hall,BD
为了确定酵母RNA聚合酶III第二大亚基中具有催化作用的重要氨基酸变化，我们对其基因的特定区域(RET1)进行了诱变，并设计了体内检测该酶增加和减少转录终止的方法。使用突变的SUP4-otRNA基因作为报告基因，其中一例过早终止，另一例未能终止，我们分别筛选了转录终止减少和增加的突变RET1文库。在两种情况下，抑制表型的增加都被评为减少了携带ade2-1ochre突变的酵母菌株中红色素的积累。在RET1基因编码蛋白质的300到325、455到486、487到521和1061到1082氨基酸区域获得了终止性改变突变。在氨基酸序列的保守程度上，从第一个区域的高度可变到最后两个区域的高度保守。残基300到325主要产生末端减少的突变体，而在1061到1082区域只获得末端增加的突变体。所有的突变都恢复了，同时导致了一个SUP4等位基因的抑制增加，导致了另一个等位基因的抑制减少，从而证实了表型是由于改变的终止而不是转录启动水平的提高。用几个强突变体的提取物进行的体外转录反应表明，突变聚合酶对RNA终止子序列的反应方式与其体内终止表型相匹配。
In order to identify catalytically important amino acid changes within the second-largest subunit of yeast RNA polymerase III, we mutagenized selected regions of its gene (RET1) and devised in vivo assays for both increased and decreased transcription termination by this enzyme. Using as the reporter gene a mutantSUP4-otRNA gene that in one case terminates prematurely and in the other case fails to terminate, we screened mutagenizedRET1libraries for reduced and increased transcription termination, respectively. The gain in suppression phenotype was in both cases scored as a reduction in the accumulation of red pigment in yeast strains harboring theade2-1ochre mutation. Termination-altering mutations were obtained in regions of theRET1gene encoding amino acids 300 to 325, 455 to 486, 487 to 521, and 1061 to 1082 of the protein. In degree of amino acid sequence conservation, these range from highly variable in the first to highly conserved in the last two regions. Residues 300 to 325 yielded mainly reduced-termination mutants, while in region 1061 to 1082, increased-termination mutants were obtained exclusively. All mutants recovered, while causing gain of suppression with oneSUP4allele, brought about a reduction in suppression with the other allele, thus confirming that the phenotype is due to altered termination rather than an elevated level of transcription initiation. In vitro transcription reactions performed with extracts from several strong mutants demonstrated that the mutant polymerases respond to RNA terminator sequences in a manner that matches their in vivo termination phenotypes.