Rossignol M, Kolb-Cheynel I, Egly J M
Institut de Génétique et de Biologie Moléculaire et Cellulaire, UPR 6520 (CNRS), Unité 184 (INSERM), Illkirch, CU de Strasbourg, France.
EMBO J. 1997 Apr 1;16(7):1628-37. doi: 10.1093/emboj/16.7.1628.
The transcription/DNA repair factor TFIIH consists of nine subunits, several exhibiting known functions: helicase/ATPase, kinase activity and DNA binding. Three subunits of TFIIH, cdk7, cyclin H and MAT1, form a ternary complex, cdk-activating kinase (CAK), found either on its own or as part of TFIIH. In the present work, we demonstrate that purified human CAK complex (free CAK) and recombinant CAK (rCAK) produced in insect cells exhibit a strong preference for the cyclin-dependent kinase 2 (cdk2) over a ctd oligopeptide substrate (which mimics the carboxy-terminal domain of the RNA polymerase II). In contrast, TFIIH preferentially phosphorylates the ctd as well as TFIIE alpha, but not cdk2. TFIIH was resolved into four subcomplexes: the kinase complex composed of cdk7, cyclin H and MAT1; the core TFIIH which contains XPB, p62, p52, p44 and p34; and two other subcomplexes in which XPD is found associated with either the kinase complex or with the core TFIIH. Using these fractions, we demonstrate that TFIIH lacking the CAK subcomplex completely recovers its transcriptional activity in the presence of free CAK. Furthermore, studies examining the interactions between TFIIH subunits provide evidence that CAK is integrated within TFIIH via XPB and XPD.
转录/DNA修复因子TFIIH由九个亚基组成，其中几个具有已知功能：解旋酶/ATP酶、激酶活性和DNA结合。TFIIH的三个亚基，即细胞周期蛋白依赖性激酶7（cdk7）、细胞周期蛋白H（cyclin H）和MAT1，形成一个三元复合物，即细胞周期蛋白依赖性激酶激活激酶（CAK），它可以单独存在，也可以作为TFIIH的一部分存在。在本研究中，我们证明，纯化的人CAK复合物（游离CAK）和在昆虫细胞中产生的重组CAK（rCAK）对细胞周期蛋白依赖性激酶2（cdk2）的偏好远高于对一个CTD寡肽底物（模拟RNA聚合酶II的羧基末端结构域）的偏好。相比之下，TFIIH优先磷酸化CTD以及TFIIEα，但不磷酸化cdk2。TFIIH被分解为四个亚复合物：由cdk7、细胞周期蛋白H和MAT1组成的激酶复合物；包含XPB、p62、p52、p44和p34的核心TFIIH；以及另外两个亚复合物，其中XPD与激酶复合物或核心TFIIH相关联。利用这些组分，我们证明，在游离CAK存在的情况下，缺乏CAK亚复合物的TFIIH完全恢复了其转录活性。此外，对TFIIH亚基之间相互作用的研究提供了证据，表明CAK通过XPB和XPD整合到TFIIH中。
