Eukaryotic transcription requires the assembly of a multisubunit preinitiation complex (PIC) composed of RNA polymerase II (Pol II) and the general transcription factors. The coactivator Mediator is recruited by transcription factors, facilitates the assembly of the PIC, and stimulates phosphorylation of the Pol II C-terminal domain (CTD) by the TFIIH subunit CDK7. Here, we present the cryo–electron microscopy structure of the human Mediator-bound PIC at a resolution below 4 angstroms. Transcription factor binding sites within Mediator are primarily flexibly tethered to the tail module. CDK7 is stabilized by multiple contacts with Mediator. Two binding sites exist for the Pol II CTD, one between the head and middle modules of Mediator and the other in the active site of CDK7, providing structural evidence for Pol II CTD phosphorylation within the Mediator-bound PIC.

真核转录需要由 RNA 聚合酶 II (Pol II) 和通用转录因子组成的多亚基预起始复合物 (PIC) 的组装。共激活介体由转录因子募集，促进 PIC 的组装，并刺激 TFIIH 亚基 CDK7 对 Pol II C 末端结构域 (CTD) 的磷酸化。在这里，我们展示了分辨率低于 4 埃的人类介体结合 PIC 的冷冻电子显微镜结构。介体内的转录因子结合位点主要灵活地连接到尾部模块。CDK7 通过与 Mediator 的多次接触而稳定。Pol II CTD 存在两个结合位点，一个位于 Mediator 的头部和中间模块之间，另一个位于 CDK7 的活性位点，为 Pol II CTD 在 Mediator 结合的 PIC 内磷酸化提供了结构证据。