Article

TFIIH generates a six-base-pair open complex during RNAP II transcription initiation and start-site scanning

  • Nature Structural & Molecular Biology volume 24, pages 11391145 (2017)
  • doi:10.1038/nsmb.3500
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Abstract

Eukaryotic mRNA transcription initiation is directed by the formation of the megadalton-sized preinitiation complex (PIC). After PIC formation, double-stranded DNA (dsDNA) is unwound to form a single-stranded DNA bubble, and the template strand is loaded into the polymerase active site. DNA opening is catalyzed by Ssl2 (XPB), the dsDNA translocase subunit of the basal transcription factor TFIIH. In yeast, transcription initiation proceeds through a scanning phase during which downstream DNA is searched for optimal start sites. Here, to test models for initial DNA opening and start-site scanning, we measure the DNA-bubble sizes generated by Saccharomyces cerevisiae PICs in real time using single-molecule magnetic tweezers. We show that ATP hydrolysis by Ssl2 opens a 6-base-pair (bp) bubble that grows to 13 bp in the presence of NTPs. These observations support a two-step model wherein ATP-dependent Ssl2 translocation leads to a 6-bp open complex that RNA polymerase II expands via NTP-dependent RNA transcription.

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Acknowledgements

The research was supported by the National Science Foundation Molecular and Cellular Biosciences Grant 1243918 (to E.A.G.) and by National Institutes of Health General Medical Science Grants 5R01GM120559 (to E.A.G.) and 2R01GM053451 (to S.H.). E.J.T. was partially supported by a Keck Postdoctoral Fellowship.

Author information

Affiliations

  1. Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, Missouri, USA.

    • Eric J Tomko
    •  & Eric A Galburt
  2. Division of Basic Sciences, Fred Hutchinson Cancer Research Center, Seattle, Washington, USA.

    • James Fishburn
    •  & Steven Hahn

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Contributions

Author contributions were as follows: E.J.T., J.F., S.H., and E.A.G. designed the research. E.J.T. performed the magnetic tweezers experiments. J.F. purified factors and performed the in vitro transcription reactions. E.J.T. and E.A.G. analyzed single-molecule data. J.F. and S.H. analyzed in vitro experiments. E.J.T., J.F., S.H., and E.A.G. wrote the paper.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Eric A Galburt.

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