RNA POLYMERASE-II SUBUNIT COMPOSITION, STOICHIOMETRY, AND PHOSPHORYLATION
RNA POLYMERASE-II SUBUNIT COMPOSITION, STOICHIOMETRY, AND PHOSPHORYLATION
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DOI：
10.1128/mcb.10.5.1915
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发表时间：
1990-05-01
影响因子：
5.3
通讯作者：
YOUNG, RA
中科院分区：
文献类型：
--
作者：
KOLODZIEJ, PA;WOYCHIK, N;YOUNG, RA
通过将表位编码序列连接到表征良好的RNA聚合酶II亚基基因（RPB3）上，并通过免疫沉淀该基因及其相关多肽的产物，研究了酿酒酵母中RNA聚合酶II亚基的组成、化学计量学和磷酸化。免疫纯化酶催化。-amanitin-sensitive RNA的体外合成。免疫沉淀的10个多肽在大小和数量上与先前描述的RNA聚合酶II用常规柱层析纯化的多肽相同。亚基的相对化学计量是根据亚基序列的知识和用[35S]蛋氨酸标记的程度推断出来的。32p标记细胞提取物的免疫沉淀显示，RPB1、RPB2和RPB6这三个亚基在体内被磷酸化。可以区分RPB1的磷酸化和非磷酸化形式；大约一半的RNA聚合酶II分子含有磷酸化的RPB1亚基。这些结果更精确地定义了真核生物RNA聚合酶II的亚基组成和磷酸化。
RNA polymerase II subunit composition, stoichiometry, and phosphorylation were investigated in Saccharomyces cerevisiae by attaching an epitope coding sequence to a well-characterized RNA polymerase II subunit gene (RPB3) and by immunoprecipitating the product of this gene with its associated polypeptides. The immunopurified enzyme catalyzed .alpha.-amanitin-sensitive RNA synthesis in vitro. The 10 polypeptides that immunoprecipitated were identical in size and number to those previously described for RNA polymerase II purified by conventional column chromatography. The relative stoichiometry of the subunits was deduced from knowledge of the sequence of the subunits and from the extent of labeling with [35S]methionine. Immunoprecipitation from 32P-labeled cell extracts revealed that three of the subunits, RPB1, RPB2, and RPB6, are phosphorylated in vivo. Phosphorylated and unphosphorylated forms of RPB1 could be distinguished; approximately half of the RNA polymerase II molecules contained a phosphorylated RPB1 subunit. These results more precisely define the subunit composition and phosphorylation of a eucaryotic RNA polymerase II enzyme.