Suppressor analysis of temperature-sensitive RNA polymerase I mutations in Saccharomyces cerevisiae: suppression of mutations in a zinc-binding motif by transposed mutant genes.
Suppressor analysis of temperature-sensitive RNA polymerase I mutations in Saccharomyces cerevisiae: suppression of mutations in a zinc-binding motif by transposed mutant genes.
复制标题
酿酒酵母温度敏感 RNA 聚合酶 I 突变的抑制分析：通过转置突变基因抑制锌结合基序的突变。
DOI：
10.1128/mcb.11.2.746-753.1991
复制
发表时间：
1991
影响因子：
5.3
通讯作者：
Nomura,M
中科院分区：
文献类型：
--
作者：
McCusker,JH;Yamagishi,M;Kolb,JM;Nomura,M
从酿酒酵母（Saccharomyces cerevisiae）的两个温度敏感突变体（rpa190-1和rpa190-5）开始，这两个突变体都是RNA聚合酶I最大亚基（A190）的锌结合结构域的氨基酸取代，我们分离了许多携带rpa190突变的外基因抑制子（SRP）的独立假逆转物。所有这些突变都比相应的野生型基因显性。通过将每个抑制突变体与所有其他抑制突变体杂交并分析分离，他们被分类到至少7个不同的位点。研究了代表这7个基因座的SRPmutations对其他已知nrpa190突变的影响。所有的基因突变都能抑制thrpa190-1和rpa190-5。此外，一个特定的抑制因子SRP5被发现抑制另外两个rpa190突变以及一个rpa190缺失。Southern blot分析结合遗传杂交表明，srp5定位于XV染色体上松散连接的torpa190区域，并在两个拷贝中代表转置突变基因。利用抗A190抗血清对A190亚基进行分析表明，在rpa190-1突变体中，当温度升高至37°C时，A190的细胞浓度下降，RNA聚合酶I也随之下降，而在携带srp5的突变株中，这种下降得到了部分缓解，可能是由于基因剂量增加导致合成增加。这些结果表明，无论锌结合结构域是否直接参与组装酶与DNA的相互作用，锌结合结构域在酶的组装和/或稳定性所必需的蛋白质-蛋白质相互作用中起着重要作用。
Starting with two temperature-sensitive mutants (rpa190-1andrpa190-5) of Saccharomyces cerevisiae both of which are amino acid substitutions in the putative zinc-binding domain of the largest subunit (A190) of RNA polymerase I, we have isolated many independent pseudorevertants carrying extragenic suppressors (SRP) ofrpa190mutations. All theSRPmutations were dominant over the corresponding wild-type genes. They were classified into at least seven different loci by crossing each suppressed mutant with all of the other suppressed mutants and analyzing segregants.SRPmutations representing each of the seven loci were studied for their effects on other knownrpa190mutations. All of theSRPmutations were able to suppress bothrpa190-1andrpa190-5. In addition, one particular suppressor, SRP5 was found to suppress two otherrpa190mutations as well as anrpa190deletion. Southern blot analysis combined with genetic crosses demonstrated thatSRP5maps to a region on chromosome XV loosely linked torpa190and represents a transposed mutant gene in two copies. Analysis of the A190 subunit by using anti-A190 antiserum indicated that the cellular concentration of A190 and hence of RNA polymerase I decreases inrpa190-1mutants after a shift to 37°C and that in the mutant strain carryingSRP5this decrease is partially alleviated, presumably because of increased synthesis caused by increased gene dosage. These results suggest that the zinc-binding domain plays an important role in protein-protein interaction essential for the assembly and/or stability of the enzyme, regardless of whether it also participates directly in the interaction of the assembled enzyme with DNA.