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A DNA-tethered cleavage probe reveals the path for promoter DNA in the yeast preinitiation complex

Abstract

To directly map the position of promoter DNA within the RNA polymerase II (Pol II) transcription preinitiation complex (PIC), FeBABE was tethered to specific sites within the HIS4 promoter and used to map exposed surfaces of Pol II and the general transcription factors in proximity to DNA. Our results distinguish between previously proposed models for PIC structure and demonstrate that downstream promoter DNA is positioned over the central cleft of Pol II, with DNA upstream of TATA extending toward the Pol II subunit Rpb3. Also mapped were segments of TFIIB, TFIIE, TFIIF and TFIIH in proximity to promoter DNA. DNA downstream of the transcription bubble maps to a path between the two helicase subdomains of the TFIIH subunit Rad25 (also called XPB). Together, our results show how the general factors and Pol II converge on promoter DNA within the PIC.

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Figure 1: HIS4 promoter templates tethered to FeBABE support transcription.
Figure 2: Directed hydroxyl radical probing of Pol II subunit Rpb1.
Figure 3: Directed hydroxyl radical probing of Pol II subunit Rpb2.
Figure 4: Model for the path of promoter DNA in the PIC.
Figure 5: Hydroxyl radical probing of TFIIH subunit Rad25.
Figure 6: Directed hydroxyl radical probing of TFIIF subunits Tfg1 and Tfg2.
Figure 7: Directed hydroxyl radical probing of TFIIE subunits Tfa1 and Tfa2.
Figure 8: Summary of hydroxyl radical cleavage of Pol II and GTFs in the PIC.

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Acknowledgements

We thank R. Ebright for the suggestion of the scrunching mechanism for yeast Pol II, H.T. Chen and L. Warfield (Fred Hutchinson Cancer Research Center) for yeast strains and nuclear extracts, and L. Fan and J. Tainer for providing the coordinates of A. fulgidus XPB before public release. We thank B. Schmidt and J. Roe for technical advice on efficient conjugation of FeBABE to DNA and H.T. Chen, N. Mohibullah, B. Moorefield and I. Whitehouse for discussions and comments on the manuscript. This work was supported by grant 5RO1GM053451 from the US National Institutes of Health. G. Miller was supported in part as a postdoctoral associate of the Howard Hughes Medical Institute.

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Correspondence to Steven Hahn.

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Supplementary information

Supplementary Fig. 1

Directed hydroxyl radical probing of RNA polymerase II subunits Rpb1 and Rpb2 with FeBABE attached to the template strand (PDF 337 kb)

Supplementary Fig. 2

In vitro–translated peptide fragments of Rpb1 and Rpb2 used as molecular standards for calculating FeBABE cleavage sites (PDF 617 kb)

Supplementary Fig. 3

In vitro–translated peptide fragments of Sua7, Rad25, Tfg1, Tfg2, Tfa1 and Tfa2 used as molecular standards for calculating FeBABE cleavage sites (PDF 1196 kb)

Supplementary Fig. 4

Directed hydroxyl radical probing of TFIIB in the PIC (PDF 156 kb)

Supplementary Fig. 5

Directed hydroxyl radical probing of TFIIH subunit Rad25 and TFIIF subunits Tfg1 and Tfg2 with FeBABE attached to the template strand (PDF 448 kb)

Supplementary Fig. 6

Rad25/XPB alignment used in structure modeling of the Rad25 helicase domains (PDF 53 kb)

Supplementary Table 1

Summary of RNA polymerase II and general transcription factor cleavage sites generated by FeBABE attached to HIS4 promoter DNA (PDF 33 kb)

Supplementary Methods (PDF 68 kb)

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Miller, G., Hahn, S. A DNA-tethered cleavage probe reveals the path for promoter DNA in the yeast preinitiation complex. Nat Struct Mol Biol 13, 603–610 (2006). https://doi.org/10.1038/nsmb1117

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