Cyclin-dependent kinase 7 (CDK7), Cyclin H, and the RING-finger protein MAT1 form the heterotrimeric CDK-activating kinase (CAK) complex which is vital for transcription and cell-cycle control. When associated with the general transcription factor II H (TFIIH) it activates RNA polymerase II by hyperphosphorylation of its C-terminal domain (CTD). In the absence of TFIIH the trimeric complex phosphorylates the T-loop of CDKs that control cell-cycle progression. CAK holds a special position among the CDK branch due to this dual activity and the dependence on two proteins for activation. We solved the structure of the CAK complex from the model organism Chaetomium thermophilum at 2.6-Å resolution. Our structure reveals an intricate network of interactions between CDK7 and its two binding partners MAT1 and Cyclin H, providing a structural basis for the mechanism of CDK7 activation and CAK activity regulation. In vitro activity measurements and functional mutagenesis show that CDK7 activation can occur independent of T-loop phosphorylation and is thus exclusively MAT1-dependent by positioning the CDK7 T-loop in its active conformation.

细胞周期蛋白依赖性激酶7（CDK7），细胞周期蛋白H和RING指蛋白MAT1形成异三聚体CDK激活激酶（CAK）复合物，这对转录和细胞周期控制至关重要。当与一般转录因子II H（TFIIH）相关时，它会通过其C末端结构域（CTD）的过度磷酸化来激活RNA聚合酶II。在没有TFIIH的情况下，三聚体复合物使控制细胞周期进程的CDK的T环磷酸化。由于这种双重活性和对两种蛋白质的激活依赖性，CAK在CDK分支中占有特殊的位置。我们从模型生物嗜热Chaetomium thermophilum解开了CAK复合物的结构分辨率为2.6Å。我们的结构揭示了CDK7及其两个结合伴侣MAT1和Cyclin H之间复杂的相互作用网络，为CDK7激活和CAK活性调节的机制提供了结构基础。体外活性测量和功能诱变表明，CDK7激活可以独立于T环磷酸化而发生，因此通过将CDK7 T环定位在其活性构象中，它仅依赖于MAT1。