RESUMO
Transcription of eukaryotic protein-coding genes begins with assembly of the RNA polymerase (Pol) II initiation complex and promoter DNA opening. Here we report cryo-electron microscopy (cryo-EM) structures of yeast initiation complexes containing closed and open DNA at resolutions of 8.8 Å and 3.6 Å, respectively. DNA is positioned and retained over the Pol II cleft by a network of interactions between the TATA-box-binding protein TBP and transcription factors TFIIA, TFIIB, TFIIE, and TFIIF. DNA opening occurs around the tip of the Pol II clamp and the TFIIE 'extended winged helix' domain, and can occur in the absence of TFIIH. Loading of the DNA template strand into the active centre may be facilitated by movements of obstructing protein elements triggered by allosteric binding of the TFIIE 'E-ribbon' domain. The results suggest a unified model for transcription initiation with a key event, the trapping of open promoter DNA by extended protein-protein and protein-DNA contacts.
Assuntos
DNA/metabolismo , DNA/ultraestrutura , Complexos Multiproteicos/química , Complexos Multiproteicos/ultraestrutura , Conformação de Ácido Nucleico , Regiões Promotoras Genéticas , Iniciação da Transcrição Genética , Sítio Alostérico , Sequência de Bases , Microscopia Crioeletrônica , DNA/química , Modelos Biológicos , Dados de Sequência Molecular , Movimento , Complexos Multiproteicos/metabolismo , Ligação Proteica , Estrutura Terciária de Proteína , RNA Polimerase II/química , RNA Polimerase II/metabolismo , RNA Polimerase II/ultraestrutura , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/ultraestrutura , Proteína de Ligação a TATA-Box/química , Proteína de Ligação a TATA-Box/metabolismo , Proteína de Ligação a TATA-Box/ultraestrutura , Moldes Genéticos , Fatores de Transcrição TFII/química , Fatores de Transcrição TFII/metabolismo , Fatores de Transcrição TFII/ultraestruturaRESUMO
The conserved co-activator complex Mediator enables regulated transcription initiation by RNA polymerase (Pol) II. Here we reconstitute an active 15-subunit core Mediator (cMed) comprising all essential Mediator subunits from Saccharomyces cerevisiae. The cryo-electron microscopic structure of cMed bound to a core initiation complex was determined at 9.7 Å resolution. cMed binds Pol II around the Rpb4-Rpb7 stalk near the carboxy-terminal domain (CTD). The Mediator head module binds the Pol II dock and the TFIIB ribbon and stabilizes the initiation complex. The Mediator middle module extends to the Pol II foot with a 'plank' that may influence polymerase conformation. The Mediator subunit Med14 forms a 'beam' between the head and middle modules and connects to the tail module that is predicted to bind transcription activators located on upstream DNA. The Mediator 'arm' and 'hook' domains contribute to a 'cradle' that may position the CTD and TFIIH kinase to stimulate Pol II phosphorylation.