Kolodziej P A, Woychik N, Liao S M, Young R A
Whitehead Institute for Biomedical Research, Nine Cambridge Center, Massachusetts 02142.
Mol Cell Biol. 1990 May;10(5):1915-20. doi: 10.1128/mcb.10.5.1915-1920.1990.
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.
通过将一个表位编码序列连接到一个已充分表征的RNA聚合酶II亚基基因（RPB3）上，并通过用其相关多肽免疫沉淀该基因的产物，对酿酒酵母中的RNA聚合酶II亚基组成、化学计量和磷酸化进行了研究。免疫纯化的酶在体外催化α-鹅膏蕈碱敏感的RNA合成。免疫沉淀的10种多肽在大小和数量上与先前通过常规柱色谱纯化的RNA聚合酶II所描述的多肽相同。亚基的相对化学计量是根据亚基的序列知识和[35S]甲硫氨酸的标记程度推导出来的。从32P标记的细胞提取物中进行免疫沉淀显示，三个亚基RPB1、RPB2和RPB6在体内被磷酸化。可以区分RPB1的磷酸化和未磷酸化形式；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RNA聚合酶II酶的亚基组成和磷酸化。
