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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References
Hahn, S. Structure and mechanism of the RNA polymerase II transcription machinery. Nat. Struct. Mol. Biol. 11, 394–403 (2004).
Giardina, C. & Lis, J.T. DNA melting on yeast RNA polymerase II promoters. Science 261, 759–762 (1993).
Pinto, I., Wu, W.H., Na, J.G. & Hampsey, M. Characterization of sua7 mutations defines a domain of TFIIB involved in transcription start site selection in yeast. J. Biol. Chem. 269, 30569–30573 (1994).
Sun, Z.W. & Hampsey, M. Identification of the gene (SSU71/TFG1) encoding the largest subunit of transcription factor TFIIF as a suppressor of a TFIIB mutation in Saccharomyces cerevisiae. Proc. Natl. Acad. Sci. USA 92, 3127–3131 (1995).
Chen, B.S. & Hampsey, M. Functional interaction between TFIIB and the Rpb2 subunit of RNA polymerase II: implications for the mechanism of transcription initiation. Mol. Cell. Biol. 24, 3983–3991 (2004).
Ghazy, M.A., Brodie, S.A., Ammerman, M.L., Ziegler, L.M. & Ponticelli, A.S. Amino acid substitutions in yeast TFIIF confer upstream shifts in transcription initiation and altered interaction with RNA polymerase II. Mol. Cell. Biol. 24, 10975–10985 (2004).
Freire-Picos, M.A., Krishnamurthy, S., Sun, Z.W. & Hampsey, M. Evidence that the Tfg1/Tfg2 dimer interface of TFIIF lies near the active center of the RNA polymerase II initiation complex. Nucleic Acids Res. 33, 5045–5052 (2005).
Chen, H.T. & Hahn, S. Binding of TFIIB to RNA polymerase II: mapping the binding site for the TFIIB zinc ribbon domain within the preinitiation complex. Mol. Cell 12, 437–447 (2003).
Chen, H.T. & Hahn, S. Mapping the location of TFIIB within the RNA polymerase II transcription preinitiation complex: a model for the structure of the PIC. Cell 119, 169–180 (2004).
Bushnell, D.A., Westover, K.D., Davis, R.E. & Kornberg, R.D. Structural basis of transcription: an RNA polymerase II–TFIIB cocrystal at 4.5 Angstroms. Science 303, 983–988 (2004).
Sun, Z.W., Tessmer, A. & Hampsey, M. Functional interaction between TFIIB and the Rpb9 (Ssu73) subunit of RNA polymerase II in Saccharomyces cerevisiae. Nucleic Acids Res. 24, 2560–2566 (1996).
Ziegler, L.M., Khaperskyy, D.A., Ammerman, M.L. & Ponticelli, A.S. Yeast RNA polymerase II lacking the Rpb9 subunit is impaired for interaction with transcription factor IIF. J. Biol. Chem. 278, 48950–48956 (2003).
Holstege, F.C., Fiedler, U. & Timmers, H.T. Three transitions in the RNA polymerase II transcription complex during initiation. EMBO J. 16, 7468–7480 (1997).
Dvir, A. Promoter escape by RNA polymerase II. Biochim. Biophys. Acta 1577, 208–223 (2002).
Pal, M., Ponticelli, A.S. & Luse, D.S. The role of the transcription bubble and TFIIB in promoter clearance by RNA polymerase II. Mol. Cell 19, 101–110 (2005).
Cramer, P. et al. Architecture of RNA polymerase II and implications for the transcription mechanism. Science 288, 640–649 (2000).
Bushnell, D.A. & Kornberg, R.D. Complete, 12-subunit RNA polymerase II at 4.1-A resolution: implications for the initiation of transcription. Proc. Natl. Acad. Sci. USA 100, 6969–6973 (2003).
Armache, K.J., Kettenberger, H. & Cramer, P. Architecture of initiation-competent 12-subunit RNA polymerase II. Proc. Natl. Acad. Sci. USA 100, 6964–6968 (2003).
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).
Kim, T.K. et al. Trajectory of DNA in the RNA polymerase II transcription preinitiation complex. Proc. Natl. Acad. Sci. USA 94, 12268–12273 (1997).
Robert, F. et al. Wrapping of promoter DNA around the RNA polymerase II initiation complex induced by TFIIF. Mol. Cell 2, 341–351 (1998).
Douziech, M. et al. Mechanism of promoter melting by the xeroderma pigmentosum complementation group B helicase of transcription factor IIH revealed by protein-DNA photo-cross-linking. Mol. Cell. Biol. 20, 8168–8177 (2000).
Kim, T.K., Ebright, R.H. & Reinberg, D. Mechanism of ATP-dependent promoter melting by transcription factor IIH. Science 288, 1418–1422 (2000).
Chung, W.H. et al. RNA polymerase II/TFIIF structure and conserved organization of the initiation complex. Mol. Cell 12, 1003–1013 (2003).
Chin, J.W. et al. An expanded eukaryotic genetic code. Science 301, 964–967 (2003).
Kauer, J.C., Erickson-Viitanen, S., Wolfe, H.R. Jr. & DeGrado, W.F. p-Benzoyl-L-phenylalanine, a new photoreactive amino acid. Photolabeling of calmodulin with a synthetic calmodulin-binding peptide. J. Biol. Chem. 261, 10695–10700 (1986).
Giuliodori, S. et al. A composite upstream sequence motif potentiates tRNA gene transcription in yeast. J. Mol. Biol. 333, 1–20 (2003).
Warfield, L., Ranish, J.A. & Hahn, S. Positive and negative functions of the SAGA complex mediated through interaction of Spt8 with TBP and the N-terminal domain of TFIIA. Genes Dev. 18, 1022–1034 (2004).
Fishburn, J., Mohibullah, N. & Hahn, S. Function of a eukaryotic transcription activator during the transcription cycle. Mol. Cell 18, 369–378 (2005).
Reeves, W.M. & Hahn, S. Targets of the Gal4 transcription activator in functional transcription complexes. Mol. Cell. Biol. 25, 9092–9102 (2005).
Nikolov, D.B. et al. Crystal structure of a TFIIB-TBP-TATA-element ternary complex. Nature 377, 119–128 (1995).
Tsai, F.T. & Sigler, P.B. Structural basis of preinitiation complex assembly on human pol II promoters. EMBO J. 19, 25–36 (2000).
Hekmatpanah, D.S. & Young, R.A. Mutations in a conserved region of RNA polymerase II influence the accuracy of mRNA start site selection. Mol. Cell. Biol. 11, 5781–5791 (1991).
Gaiser, F., Tan, S. & Richmond, T.J. Novel dimerization fold of RAP30/RAP74 in human TFIIF at 1.7 A resolution. J. Mol. Biol. 302, 1119–1127 (2000).
Geiger, J.H., Hahn, S., Lee, S. & Sigler, P.B. Crystal structure of the yeast TFIIA/TBP/DNA complex. Science 272, 830–836 (1996).
Chen, H.T., Legault, P., Glushka, J., Omichinski, J.G. & Scott, R.A. Structure of a (Cys3His) zinc ribbon, a ubiquitous motif in archaeal and eucaryal transcription. Protein Sci. 9, 1743–1752 (2000).
Bushnell, D.A., Bamdad, C. & Kornberg, R.D. A minimal set of RNA polymerase II transcription protein interactions. J. Biol. Chem. 271, 20170–20174 (1996).
Berroteran, R.W., Ware, D.E. & Hampsey, M. The sua8 suppressors of Saccharomyces cerevisiae encode replacements of conserved residues within the largest subunit of RNA polymerase II and affect transcription start site selection similarly to sua7 (TFIIB) mutations. Mol. Cell. Biol. 14, 226–237 (1994).
Kapanidis, A.N. et al. Initial transcription by RNA polymerase proceeds through a DNA-scrunching mechanism. Science 314, 1144–1147 (2006).
Revyakin, A., Liu, C., Ebright, R.H. & Strick, T.R. Abortive initiation and productive initiation by RNA polymerase involve DNA scrunching. Science 314, 1139–1143 (2006).
Ranish, J.A., Yudkovsky, N. & Hahn, S. Intermediates in formation and activity of the RNA polymerase II preinitiation complex: holoenzyme recruitment and a postrecruitment role for the TATA box and TFIIB. Genes Dev. 13, 49–63 (1999).
Sambrook, J., Fritsch, E.F. & Maniatis, T. in Molecular Cloning: A Laboratory Manual 2nd edn. 15.74–15.79 (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, USA, 1989).
Westover, K.D., Bushnell, D.A. & Kornberg, R.D. Structural basis of transcription: separation of RNA from DNA by RNA polymerase II. Science 303, 1014–1016 (2004).
Nicholls, A., Sharp, K.A. & Honig, B. Protein folding and association: insights from the interfacial and thermodynamic properties of hydrocarbons. Proteins 11, 281–296 (1991).
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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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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DOI: https://doi.org/10.1038/nsmb1272
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