Liu Ying, Kung Charles, Fishburn James, Ansari Aseem Z, Shokat Kevan M, Hahn Steven
Division of Basic Sciences, Fred Hutchinson Cancer Research Center and Howard Hughes Medical Institute, Seattle, Washington 98109, USA.
Mol Cell Biol. 2004 Feb;24(4):1721-35. doi: 10.1128/MCB.24.4.1721-1735.2004.
Three cyclin-dependent kinases, CDK7, -8, and -9, are specifically involved in transcription by RNA polymerase II (Pol II) and target the Pol II C-terminal domain (CTD). The role of CDK7 and CDK8 kinase activity in transcription has been unclear, with CDK7 shown to have variable effects on transcription and CDK8 suggested to repress transcription and/or to target other gene-specific factors. Using a chemical genetics approach, the Saccharomyces cerevisiae homologs of these kinases, Kin28 and Srb10, were engineered to respond to a specific inhibitor and the inhibitor was used to test the role of these kinases in transcription in vivo and in vitro. In vitro, these kinases can both promote transcription, with up to 70% of transcription abolished when both kinases are inhibited together. Similarly, in vivo inhibition of both kinases together gives the strongest decrease in transcription, as measured by chromatin immunoprecipitation of Pol II. Kin28 and Srb10 also have overlapping roles in promoting ATP-dependent dissociation of the preinitiation complex (PIC) into the Scaffold complex. Using the engineered kinases and an ATP analog, specific kinase substrates within the PIC were identified. In addition to the previously known substrate, the Pol II CTD, it was found that Kin28 phosphorylates two subunits of Mediator and Srb10 targets two subunits of TFIID for phosphorylation.
三种细胞周期蛋白依赖性激酶，即CDK7、CDK8和CDK9，特异性地参与RNA聚合酶II（Pol II）介导的转录过程，并作用于Pol II的C末端结构域（CTD）。CDK7和CDK8激酶活性在转录中的作用一直不明确，CDK7对转录表现出多种不同影响，而CDK8则被认为可抑制转录和/或作用于其他基因特异性因子。利用化学遗传学方法，对这些激酶在酿酒酵母中的同源物Kin28和Srb10进行改造，使其对特定抑制剂产生反应，并使用该抑制剂在体内和体外测试这些激酶在转录中的作用。在体外，这些激酶均可促进转录，当两种激酶同时被抑制时，转录可被消除多达70%。同样，通过Pol II的染色质免疫沉淀法检测发现，在体内同时抑制这两种激酶会导致转录的最大程度下降。Kin28和Srb10在促进前起始复合物（PIC）依赖ATP解离为支架复合物的过程中也具有重叠作用。利用改造后的激酶和一种ATP类似物，确定了PIC内的特异性激酶底物。除了先前已知的底物Pol II CTD外，还发现Kin28可磷酸化中介体的两个亚基，而Srb10可靶向TFIID的两个亚基进行磷酸化。
