The Cdk8 kinase module (CKM) in Mediator, comprising Med13, Med12, CycC, and Cdk8, regulates RNA polymerase II transcription through kinase-dependent and -independent functions. Numerous pathogenic mutations causative for neurodevelopmental disorders and cancer congregate in CKM subunits. However, the structure of the intact CKM and the mechanism by which Cdk8 is non-canonically activated and functionally affected by oncogenic CKM alterations are poorly understood. Here, we report a cryo–electron microscopy structure of Saccharomyces cerevisiae CKM that redefines prior CKM structural models and explains the mechanism of Med12-dependent Cdk8 activation. Med12 interacts extensively with CycC and activates Cdk8 by stabilizing its activation (T-)loop through conserved Med12 residues recurrently mutated in human tumors. Unexpectedly, Med13 has a characteristic Argonaute-like bi-lobal architecture. These findings not only provide a structural basis for understanding CKM function and pathological dysfunction, but also further impute a previously unknown regulatory mechanism of Mediator in transcriptional modulation through its Med13 Argonaute-like features.

Mediator 中的 Cdk8 激酶模块 (CKM) 由 Med13、Med12、CycC 和 Cdk8 组成，通过激酶依赖性和独立性功能调节 RNA 聚合酶 II 转录。许多导致神经发育障碍和癌症的致病突变聚集在 CKM 亚基中。然而，人们对完整 CKM 的结构以及 Cdk8 非规范激活和功能上受致癌 CKM 改变影响的机制知之甚少。在这里，我们报告了酿酒酵母的冷冻电子显微镜结构CKM 重新定义了先前的 CKM 结构模型并解释了 Med12 依赖性 Cdk8 激活的机制。Med12 与 CycC 广泛相互作用，并通过在人类肿瘤中反复突变的保守 Med12 残基稳定其激活 (T-) 环，从而激活 Cdk8。出乎意料的是，Med13 具有典型的类似 Argonaute 的双叶结构。这些发现不仅为理解CKM功能和病理功能障碍提供了结构基础，而且通过其Med13 Argonaute样特征进一步推测了Mediator在转录调节中先前未知的调节机制。