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Architecture of the RNA polymerase II preinitiation complex and mechanism of ATP-dependent promoter opening

Nature Structural & Molecular Biology volume 19, pages 788–796 (2012)Cite this article

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Abstract

Yeast RNA polymerase II (Pol II) general transcription factor TFIIE and the TFIIH subunit Ssl2 (yeast ortholog of mammalian XPB) function in the transition of the preinitiation complex (PIC) to the open complex. We show that the three TFIIE winged-helix (WH) domains form a heterodimer, with the Tfa1 (TFIIEα) WH binding the Pol II clamp and the Tfa2 (TFIIEβ) tandem WH domain encircling promoter DNA that becomes single-stranded in the open complex. Ssl2 lies adjacent to TFIIE, enclosing downstream promoter DNA. Unlike previous proposals, comparison of the PIC and open-complex models strongly suggests that Ssl2 promotes DNA opening by functioning as a double-stranded-DNA translocase, feeding 15 base pairs into the Pol II cleft. Right-handed threading of DNA through the Ssl2 binding groove, combined with the fixed position of upstream promoter DNA, leads to DNA unwinding and the open state.

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Figure 1: Functionally important regions in TFIIE.
Figure 2: Mapping of TFIIE-FeBABE cleavage in Pol II.
Figure 3: Cleavage of TFIIB by TFIIE-FeBABE in the PIC.
Figure 4: Position of TFIIE in the PIC.
Figure 5: Effect of mutations in the TFIIE dimerization, Pol II clamp and protrusion domains.
Figure 6: Orientation of the TFIIH subunit Ssl2 (XPB) in the PIC.
Figure 7: Protein cross-linking reveals Ssl2 interactions with TFIIE and TFIIB.
Figure 8: Comparison of the PIC and open-complex (OC) models suggests the role of Ssl2 in DNA strand opening.

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Acknowledgements

We thank J. Luo and J. Ranish (Institute for Systems Biology, Seattle, Washington) for sharing their unpublished TFIIE–cross-linking data, J. Fishburn for advice and help with TFIIE purification and transcription assays, A. Vannini for coordinates of the Rpc34 WH model, G. Smith for discussions and B. Knutson, I. Kamenova and M. Bartlett for comments on the manuscript. This work was supported by grant RO1GM053451 to S.H. and Forschungsstipendium GR 3776/2-1 of the Deutsche Forschungsgemeinschaft to S.G.

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  1. Fred Hutchinson Cancer Research Center, Seattle, Washington, USA

    Sebastian Grünberg, Linda Warfield & Steven Hahn

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  1. Sebastian Grünberg
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S.G. designed and performed all biochemical experiments except for the Bpa cross-linking, which was designed and performed by L.W.; S.G. and S.H. designed and performed Tfa1 and Tfa2 genetic assays, performed structure modeling and prepared the manuscript.

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Correspondence to Steven Hahn.

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Supplementary Figures 1–5 and Supplementary Tables 1 and 2 (PDF 3323 kb)

Supplementary Movie 1

Overall structure of the PIC and open complex models. (MOV 6594 kb)

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Grünberg, S., Warfield, L. & Hahn, S. Architecture of the RNA polymerase II preinitiation complex and mechanism of ATP-dependent promoter opening. Nat Struct Mol Biol 19, 788–796 (2012). https://doi.org/10.1038/nsmb.2334

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