Structure and Mechanism of the human Transcription Initiation Machinery

Abstract

The human Mediator complex (1.2 MDa, 26 subunits) is a general factor that helps regulate expression of perhaps all protein‐coding genes. Using cryo‐EM, we defined the structural organization of the 1.9 MDa Mediator‐pol II‐TFIIF assembly, in which Mediator was bound to the activation domain of VP16. The pol II orientation within the cryo‐EM map is entirely consistent with existing biophysical studies that determined the location of other PIC factors relative to pol II itself, allowing a structure‐based model for the entire 3.5 MDa human PIC to be proposed. Whereas TFIIF was shown to be important for stably orienting pol II within this assembly, the potential role of the activator (VP16) was not assessed. Remarkably, subsequent cryo‐EM studies of human Mediator–pol II–TFIIF complexes in which Mediator was not bound to an activator have revealed that pol II does not adopt a stable orientation in the absence of an activator. These data demonstrate that activator‐Mediator binding can control pol II orientation at the Mediator‐pol II interface and suggest that activators function in part by regulating PIC structure at the promoter. A major regulator of the Mediator–pol II interaction is the 600 kDa CDK8 module, which consists of the proteins CDK8, Cyclin C, MED12, and MED13. The human CDK8 module regulates Mediator structure and function and prevents Mediator– pol II binding. Emerging data now suggest additional, expanded roles for the human CDK8 module in regulating gene expression, which will be discussed in the context of the structural model of the human PIC.