Archambault J, Schappert K T, Friesen J D
Department of Genetics, Hospital for Sick Children, Toronto, Ontario, Canada.
Mol Cell Biol. 1990 Dec;10(12):6123-31. doi: 10.1128/mcb.10.12.6123-6131.1990.
RNA polymerase II (RNAPII) is a complex multisubunit enzyme responsible for the synthesis of pre-mRNA in eucaryotes. The enzyme is made of two large subunits associated with at least eight smaller polypeptides, some of which are common to all three RNA polymerase species. We have initiated a genetic analysis of RNAPII by introducing mutations in RPO21, the gene encoding the largest subunit of RNAPII in Saccharomyces cerevisiae. We have used a yeast genomic library to isolate plasmids that can suppress a temperature-sensitive mutation in RPO21 (rpo21-4), with the goal of identifying gene products that interact with the largest subunit of RNAPII. We found that increased expression of wild-type RPO26, a single-copy, essential gene encoding a 155-amino-acid subunit common to RNAPI, RNAPII, and RNAPIII, suppressed the rpo21-4 temperature-sensitive mutation. Mutations were constructed in vitro that resulted in single amino acid changes in the carboxy-terminal portion of the RPO26 gene product. One temperature-sensitive mutation, as well as some mutations that did not by themselves generate a phenotype, were lethal in combination with rpo21-4. These results support the idea that the RPO26 and RPO21 gene products interact.
RNA聚合酶II（RNAPII）是一种负责在真核生物中合成前体mRNA的复杂多亚基酶。该酶由两个大亚基和至少八个较小的多肽组成，其中一些多肽是所有三种RNA聚合酶共有的。我们通过在酿酒酵母中编码RNAPII最大亚基的基因RPO21中引入突变，启动了对RNAPII的遗传分析。我们使用酵母基因组文库分离能够抑制RPO21（rpo21-4）温度敏感突变的质粒，目的是鉴定与RNAPII最大亚基相互作用的基因产物。我们发现，野生型RPO26（一个单拷贝的必需基因，编码RNAPI、RNAPII和RNAPIII共有的155个氨基酸的亚基）的表达增加可抑制rpo21-4温度敏感突变。在体外构建了导致RPO26基因产物羧基末端部分单个氨基酸变化的突变。一种温度敏感突变以及一些本身不产生表型的突变与rpo21-4组合时是致死的。这些结果支持RPO26和RPO21基因产物相互作用的观点。
