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

Nature Structural & Molecular Biology volume 24, pages 1139–1145 (2017)Cite this article

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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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Figure 1: Experimental setup.
Figure 2: NTP-catalyzed DNA opening.
Figure 3: Formation of stable elongation complexes.
Figure 4: ATP-catalyzed DNA opening.
Figure 5: A partially open complex is an intermediate in transcription initiation.
Figure 6: A 6-bp bubble is sufficient to support initiation in the absence of TFIIH.
Figure 7: Models of transcription initiation.

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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.

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Authors and 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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  1. Eric J Tomko
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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.

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Correspondence to Eric A Galburt.

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Integrated supplementary information

Supplementary Figure 1 Bubble size distribution of a subset of NTP traces.

Probability distribution generated from the subset of traces showing sharp open/close transitions (N = 7) collected in the presence of 500 μM NTP. The data (blue circles) are well fit (red) with only two states: a closed conformation (grey) and an open conformation with a 13 bp DNA bubble (magenta). The distribution from DNA only traces is shown for comparison (dashed line).

Supplementary Figure 2 Comparison of data collected in the presence of 500 μM ATP on negatively and positively supercoiled templates.

(a) Examples of DNA unwinding in the presence of 500 μM ATP on negatively (green) and positively (red) supercoiled templates. The changes in extension under different supercoiling directions are similar and in opposite directions suggesting that there is no large compaction occurring during DNA unwinding. (b) The distributions of length changes for negatively supercoiled templates (green) and positively supercoiled templates (red) as compared to the DNA only control (black dashed).

Supplementary Figure 3 Traces in the absence of ATP do not show DNA unwinding.

(a) The DNA extension vs. time of negatively supercoiled DNA (light grey) followed by flowing in PIC components in the absence of ATP (dark grey) and the resulting trace. (b) Two traces in the presence of PIC components and the absence of ATP under both negatively and positively supercoiled conditions. The rotation-extension curve from rotating the magnets to convert from negative to positive super-helicity takes place in the shaded regions. (c) Distributions of observed extensions of DNA alone (dashed) and in the presence of PIC components from the trace shown in (a).

Supplementary Figure 4 Comparison of data collected in the presence of 500 μM dATP and 50 μM NTP on negatively and positively supercoiled templates.

The distributions of length changes for negatively supercoiled templates (green) and positively supercoiled templates (red) as compared to the DNA only control (black dashed). The changes in extension under different supercoiling directions are similar and in opposite directions suggesting that there is no large compaction occurring during DNA unwinding.

Supplementary Figure 5 Sub1 and TFIIA do not lead to larger bubbles.

Bubble size distributions in the presence (dotted line, N = 12) and absence of Sub1 and TFIIA. Between 28 and 100 nM Sub1 and 100 nM TFIIA were introduced to the flow cell concomitantly with the general transcription factors under conditions of 500 μM dATP and 50 μM NTP as described in the text.

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Tomko, E., Fishburn, J., Hahn, S. et al. TFIIH generates a six-base-pair open complex during RNAP II transcription initiation and start-site scanning. Nat Struct Mol Biol 24, 1139–1145 (2017). https://doi.org/10.1038/nsmb.3500

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