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Molecular Visualization of the Eukaryotic Transcription Initiation Process using Cryo‐EM
Author(s) -
Nogales Eva,
Louder Robert,
He Yuan
Publication year - 2016
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.30.1_supplement.247.1
Subject(s) - transcription factor ii a , transcription factor ii h , transcription factor ii b , tata box , general transcription factor , transcription factor ii e , transcription preinitiation complex , transcription factor ii f , biology , microbiology and biotechnology , eukaryotic transcription , transcription factor ii d , transcription (linguistics) , promoter , genetics , gene , gene expression , linguistics , philosophy
The critical step of transcription initiation requires the assembly of the general transcription factors into a pre‐initiation complex (PIC) that ensures the accurate loading of Pol II at the transcription start site, carries out the opening of the DNA duplex, and facilitates clearance of the promoter by Pol II. We are using single particle cryo‐EM to investigate the assembly, architecture and structural rearrangements of the human PIC during the process of transcription initiation. Our efforts have concentrated on studying, on one hand, the general transcription factor TFIID and its recognition of core promoter DNA, and on the other a simplified PIC using TATA‐binding protein (TBP) as a surrogate for TFIID. Using an in vitro reconstitution system we visualized the step by step assembly of the TBP‐based human PIC, ultimately containing TBP, TFIIA, TFIIB, Pol II, TFIIF, TFIIE and TFIIH on promoter DNA. Our cryo‐EM structures provide pseudo‐atomic models that illuminate critical protein‐protein and protein‐DNA interactions with the PIC. Comparison of the closed PIC with the open complex has allowed us to shed light onto the process of promoter melting. The apparent movement of downstream DNA, together with the positioning of XPB, support the previously proposed translocase model for TFIIH. We propose that XPB would push and rotate the DNA with respect to a contact point of Pol II and the DNA, just upstream of the active site of XPB. These actions of XPB against the stably bound upstream DNA around the TATA box would in turn induce negative supercoiling near the transcription start site. TFIID plays a central role in the initiation of RNA polymerase II‐dependent transcription by nucleating PIC assembly at the core promoter. TFIID comprises TBP and 13 TBP‐associated factors (TAF1‐13), which specifically interact with a variety of core promoter DNA sequences. Using single‐particle cryo‐electron microscopy, we determined that TFIID exist in multiple conformational states. Two major conformers, which we refer to as the canonical and rearranged states, coexist in the apo complex. Only the rearranged state is capable of binding core promoter DNA. Recently we have determined the structure of human TFIID in complex with TFIIA and core promoter DNA at sub‐nanometer resolution. Our structure shows how all the super core promoter elements (TATA, INR, MTE and DPE) are contacted by subunits of TFIID. Together with the cryo‐EM reconstruction of a fully‐assembled human TAF‐less PIC, our structures provide novel insights into the general role of TFIID in promoter recognition, PIC assembly, and transcription initiation. Support or Funding Information This work was funded by NIGMS and HHMI.