Abstract
Eukaryotic transcription initiation requires the assembly of general transcription factors into a pre-initiation complex that ensures the accurate loading of RNA polymerase II (Pol II) at the transcription start site. The molecular mechanism and function of this assembly have remained elusive due to lack of structural information. Here we have used an in vitro reconstituted system to study the stepwise assembly of human TBP, TFIIA, TFIIB, Pol II, TFIIF, TFIIE and TFIIH onto promoter DNA using cryo-electron microscopy. Our structural analyses provide pseudo-atomic models at various stages of transcription initiation that illuminate critical molecular interactions, including how TFIIF engages Pol II and promoter DNA to stabilize both the closed pre-initiation complex and the open-promoter complex, and to regulate start--initiation complexes, combined with the localization of the TFIIH helicases XPD and XPB, support a DNA translocation model of XPB and explain its essential role in promoter opening.
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Protein Data Bank
Data deposits
Cryo-EM density maps have been deposited in the Electron Microscopy Data Bank (EMDB) under accession numbers EMD-2304 (TBP–TFIIA– TFIIB–DNA–Pol II), EMD-2305 (TBP–TFIIA–TFIIB–DNA–Pol II–TFIIF), EMD-2306 (TBP– TFIIA–TFIIB–DNA–Pol II–TFIIF–TFIIE) and EMD-2307 (TBP–TFIIA–TFIIB–DNA–Pol II– TFIIF–TFIIE in the OC mimic state). Negative stain EM density maps have been assigned accession numbers EMD-2308 (TBP–TFIIA–TFIIB–DNA–Pol II–TFIIF–TFIIE–TFIIH) and EMD-2309 (apo TFIIH).
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Acknowledgements
We thank C. Inouye for providing us with recombinant TFIIF and TFIIE; P. Grob and T. Houweling for electron microscopy and computer support, respectively; T. Goddard for help with Chimera; and members of the Nogales laboratory for technical advice on image processing. We are thankful to J. Kadonaga, J. Goodrich and M. Cianfrocco for their comments on the manuscript. We thank P. Cooper both for biochemical advice and for her comments on the manuscript. This work was funded by NIGMS (GM63072 to E.N.) and by NCI (CA127364 to D.J.T.). E.N. is a Howard Hughes Medical Institute Investigator.
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Y.H. designed and carried out the experiments; J.F. and D.J.T. provided essential reagents; Y.H. and E.N. analysed the data and wrote the paper.
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This file contains Supplementary Figures 1-16 and Supplementary References. (PDF 2461 kb)
Step-wise assembly of the human transcription PIC as revealed by successive cryo-EM reconstructions
[1] TBP (red) - TFIIA (orange) - TFIIB (blue) - DNA (green, yellow and cyan) - Pol II (grey), [2] plus TFIIF (purple), [3] plus TFIIE (maroon). Docking of available atomic coordinates leads to pseudo-atomic models of these PIC subcomplexes (in the movie shown for the last reconstruction). The negative stain reconstruction of the PIC including TFIIH (light pink) allows for the localization of the TFIIH helicases XPD (green ribbon) and XPB (blue ribbon) relative to the rest of the complex. Comparison of the reconstructions for the closed pre-initiation complex and for the open promoter complex suggests that a DNA translocation activity of XPB plays an essential role in promoter opening. (MOV 32302 kb)
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He, Y., Fang, J., Taatjes, D. et al. Structural visualization of key steps in human transcription initiation. Nature 495, 481–486 (2013). https://doi.org/10.1038/nature11991
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DOI: https://doi.org/10.1038/nature11991
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