Isolation and characterization of temperature-sensitive mutations in RPA190, the gene encoding the largest subunit of RNA polymerase I from Saccharomyces cerevisiae.
Isolation and characterization of temperature-sensitive mutations in RPA190, the gene encoding the largest subunit of RNA polymerase I from Saccharomyces cerevisiae.
复制标题
RPA190 温度敏感突变的分离和表征，RPA190 是编码酿酒酵母 RNA 聚合酶 I 最大亚基的基因。
DOI：
10.1128/mcb.8.10.3997-4008.1988
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发表时间：
1988
影响因子：
5.3
通讯作者：
Nomura,M
中科院分区：
文献类型：
--
作者：
Wittekind,M;Dodd,J;Vu,L;Kolb,JM;Buhler,JM;Sentenac,A;Nomura,M
对酿酒酵母RNA聚合酶I的温度敏感突变进行了分离和鉴定。携带该酶最大亚基编码基因RPA190的质粒经体外羟胺诱变。利用质粒改组筛选系统，分离到5种不同的质粒，它们在携带染色体中断的RPA190基因的单倍体酵母菌株中表现出温度敏感的表型。这些温度敏感等位基因被转移到染色体上的RPA190基因座，用于定位和生理实验。在不允许的温度下，所有突变菌株的RNA积累都是有缺陷的。此外，对两株突变株在37℃下的脉冲标记RNA分析表明，rRNA基因的转录减少，而5S RNA的转录相对未受影响。RNA聚合酶I是从几个在非允许温度下生长的突变菌株中部分纯化出来的，在体外测试时显示是缺陷的。对突变等位基因的精细结构作图和测序表明，所有五个突变都是独一无二的。Therpa190-1和rpa190-5突变紧密聚集在I区(S.S.Broyles和B.Moss，proc.娜塔莉。阿卡德。SCI。美国83：3141-3145,1986)，所有真核RNA聚合酶大亚基共有的锌结合区。Therpa190-3突变位于区域III和IV之间，携带该突变的菌株表现为酶合成缺陷的突变。这种突变存在于一个先前高度保守的氨基酸序列同源性的未知片段中，该片段在真核核RNA聚合酶的最大亚基中共享。另一个温度敏感突变，rpa190-2，产生了一个UGA无义密码子。
The isolation and characterization of temperature-sensitive mutations in RNA polymerase I fromSaccharomyces cerevisiaeare described. A plasmid carryingRPA190, the gene encoding the largest subunit of the enzyme, was subjected to in vitro mutagenesis with hydroxylamine. Using a plasmid shuffle screening system, five different plasmids were isolated which conferred a temperature-sensitive phenotype in haploid yeast strains carrying the disrupted chromosomalRPA190 gene. These temperature-sensitive alleles were transferred to the chromosomalRPA190 locus for mapping and physiology experiments. Accumulation of RNA was found to be defective in all mutant strains at the nonpermissive temperature. In addition, analysis of pulse-labeled RNA from two mutant strains at 37 degrees C showed that the transcription of rRNA genes was decreased, while that of 5S RNA was relatively unaffected. RNA polymerase I was partially purified from several of the mutant strains grown at the nonpermissive temperature and was shown to be deficient when assayed in vitro. Fine-structure mapping and sequencing of the mutant alleles demonstrated that all five mutations were unique. Therpa190-1 andrpa190-5 mutations are tightly clustered in region I (S.S. Broyles and B. Moss, Proc. Natl. Acad. Sci. USA 83:3141-3145, 1986), the putative zinc-binding region that is common to all eucaryotic RNA polymerase large subunits. Therpa190-3 mutation is located between regions III and IV, and a strain carrying it behaves as a mutant that is defective in the synthesis of the enzyme. This mutation lies within a previously unidentified segment of highly conserved amino acid sequence homology that is shared among the largest subunits of eucaryotic nuclear RNA polymerases. Another temperature-sensitive mutation,rpa190-2, creates a UGA nonsense codon.