RESUMO
The Mediator complex plays an essential and multi-faceted role in regulation of RNA polymerase II transcription in all eukaryotes. Structural analysis of yeast Mediator has provided an understanding of the conserved core of the complex and its interaction with RNA polymerase II but failed to reveal the structure of the Tail module that contains most subunits targeted by activators and repressors. Here we present a molecular model of mammalian (Mus musculus) Mediator, derived from a 4.0 Å resolution cryo-EM map of the complex. The mammalian Mediator structure reveals that the previously unresolved Tail module, which includes a number of metazoan specific subunits, interacts extensively with core Mediator and has the potential to influence its conformation and interactions.
Assuntos
Sequência Conservada , Mamíferos/metabolismo , Complexo Mediador/química , Complexo Mediador/metabolismo , Animais , Linhagem Celular Tumoral , Doença/genética , Complexo Mediador/ultraestrutura , Camundongos , Modelos Moleculares , Mutação/genética , Estrutura Secundária de Proteína , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/químicaRESUMO
Mediator, a large (21 polypeptides, MW â¼1 MDa) complex conserved throughout eukaryotes, plays an essential role in control of gene expression by conveying regulatory signals that influence the activity of the preinitiation complex. However, the precise mode of interaction between Mediator and RNA polymerase II (RNAPII), and the mechanism of regulation by Mediator remain elusive. We used cryo-electron microscopy and reconstituted in vitro transcription assays to characterize a transcriptionally-active complex including the Mediator Head module and components of a minimum preinitiation complex (RNAPII, TFIIF, TFIIB, TBP, and promoter DNA). Our results reveal how the Head interacts with RNAPII, affecting its conformation and function.
Assuntos
Complexo Mediador/química , RNA Polimerase II/química , RNA Polimerase II/metabolismo , Sítios de Ligação , Microscopia Crioeletrônica , Complexo Mediador/metabolismo , Complexo Mediador/ultraestrutura , Regiões Promotoras Genéticas , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Relação Estrutura-Atividade , Fatores de Transcrição TFII/química , Fatores de Transcrição TFII/metabolismoRESUMO
It is not well understood how the human Mediator complex, transcription factor IIH and RNA polymerase II (Pol II) work together with activators to initiate transcription. Activator binding alters Mediator structure, yet the functional consequences of such structural shifts remain unknown. The p53 C terminus and its activation domain interact with different Mediator subunits, and we find that each interaction differentially affects Mediator structure; strikingly, distinct p53-Mediator structures differentially affect Pol II activity. Only the p53 activation domain induces the formation of a large pocket domain at the Mediator-Pol II interaction site, and this correlates with activation of stalled Pol II to a productively elongating state. Moreover, we define a Mediator requirement for TFIIH-dependent Pol II C-terminal domain phosphorylation and identify substantial differences in Pol II C-terminal domain processing that correspond to distinct p53-Mediator structural states. Our results define a fundamental mechanism by which p53 activates transcription and suggest that Mediator structural shifts trigger activation of stalled Pol II complexes.