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The positions of TFIIF and TFIIE in the RNA polymerase II transcription preinitiation complex

Abstract

We incorporated the non-natural photoreactive amino acid p-benzoyl-L-phenylalanine (Bpa) into the RNA polymerase II (Pol II) surface surrounding the central cleft formed by the Rpb1 and Rpb2 subunits. Photo-cross-linking of preinitiation complexes (PICs) with these Pol II derivatives and hydroxyl-radical cleavage assays revealed that the TFIIF dimerization domain interacts with the Rpb2 lobe and protrusion domains adjacent to Rpb9, while TFIIE cross-links to the Rpb1 clamp domain on the opposite side of the Pol II central cleft. Mutations in the Rpb2 lobe and protrusion domains alter both Pol II–TFIIF binding and the transcription start site, a phenotype associated with mutations in TFIIF, Rpb9 and TFIIB. Together with previous biochemical and structural studies, these findings illuminate the structural organization of the PIC and the network of protein-protein interactions involved in transcription start site selection.

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Figure 1: Bpa incorporation into TFIIB and photo-cross-linking.
Figure 2: Photo-cross-linking confirms binding between the TFIIB core domain and the Rpb2 wall domain within the PIC.
Figure 3: TFIIF binds the Rpb2 lobe and protrusion domains within the PIC.
Figure 4: TFIIE interacts with the Rpb1 clamp domain within the PIC.
Figure 5: Rpb2 lobe and protrusion domain mutations alter Pol II–IIF interaction and confer an upstream shift in the transcription start site.
Figure 6: Site-specific hydroxyl-radical protein cleavage reveals the binding site for the TFIIF dimerization domain within the PIC.
Figure 7: Model of PIC assembly.

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Acknowledgements

We thank G. Miller, B. Moorefield, N. Mohibullah, T. Young and other members of the Hahn laboratory for their comments and suggestions throughout the course of this work; J. Eichner (Fred Hutchinson Cancer Research Center) for assistance with TFIIF purification; P. Schultz, J. Chin and A. Cropp (The Scripps Research Institute) for the non-natural tRNA/aminoacyl-tRNA synthetase plasmid and advice on use of non-natural amino acids; and B. Moorefield and N. Mohibullah for comments on the manuscript. This work was supported by grant 5R01GM053451 from the US National Institutes of Health to S.H.

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L.W. modified the non-natural amino acid incorporation system and performed the experiment in Figure 1. H.-T.C. performed and designed the remaining experiments. S.H. supervised the study. H.-T.C. and S.H. wrote the manuscript.

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Correspondence to Steven Hahn.

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Chen, HT., Warfield, L. & Hahn, S. The positions of TFIIF and TFIIE in the RNA polymerase II transcription preinitiation complex. Nat Struct Mol Biol 14, 696–703 (2007). https://doi.org/10.1038/nsmb1272

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