Dvir A, Stein L Y, Calore B L, Dynan W S
Department of Chemistry and Biochemistry, University of Colorado, Boulder 80309.
J Biol Chem. 1993 May 15;268(14):10440-7.
We have recently shown that a template-associated protein kinase, which phosphorylates the carboxyl-terminal domain (CTD) of RNA polymerase II, is a two-component system. We describe here the purification of these two components to apparent homogeneity from human (HeLa) cell nuclear extract. Kinase component A has a 340-kDa native molecular mass, consists of a single large polypeptide, and contains the kinase active site. Kinase component B, which is identical to the Ku autoantigen, has a 180-kDa native molecular mass, and consists of apparently equimolar 67- and 83-kDa polypeptides. Component B stimulates the activity of component A, and under some conditions, confers DNA dependence on the reaction. The purified kinase converts the CTD to the multiply phosphorylated CTD0 form. Conversion occurs processively, and this processivity is an inherent property of component A. The in vitro phosphorylated CTD0 form contains approximately equimolar phosphoserine and phosphothreonine, but no detectable phosphotyrosine.
我们最近发现，一种使RNA聚合酶II的羧基末端结构域（CTD）磷酸化的模板相关蛋白激酶是一种双组分系统。在此，我们描述了从人（HeLa）细胞核提取物中纯化这两种组分至表观均一的过程。激酶组分A的天然分子量为340 kDa，由一条单一的大多肽组成，并含有激酶活性位点。激酶组分B与Ku自身抗原相同，天然分子量为180 kDa，由明显等摩尔的67 kDa和83 kDa多肽组成。组分B刺激组分A的活性，并且在某些条件下，使反应具有DNA依赖性。纯化的激酶将CTD转化为多重磷酸化的CTD0形式。转化是连续发生的，并且这种连续性是组分A的固有特性。体外磷酸化的CTD0形式含有大致等摩尔的磷酸丝氨酸和磷酸苏氨酸，但未检测到磷酸酪氨酸。
