The long range goal of our research is the understanding of RNA polymerase II (pol II) transcription in all its aspects: initiation, RNA chain elongation, and control of the process.
Specific aims for the next project period are as follows: (1) Extension of resolution of the pol II transcribing complex structure. Preliminary results have shown the possible path of the pol II C- terminal domain (CTD), the likely role of water molecules in the protein-nucleic acid translocation process, and a rotation of the penultimate base in the DNA template that may pair with NTP prior to entry in the active center ("templated NTP"). (2) Determination of pre-initiation complex (PIC) structure. After many failed attempts at forming complexes of pol II with individual general transcription factors (GTFs), we succeeded in the assembly of a complete PIC, containing pol II and all six GTFs. This remarkable advance has opened the way to structural studies of the transcription initiation process. We propose protein-protein cross- linking and mass spectrometry (with the use of a novel ion source), soaks of pol II crystals with peptides from GTFs, and docking of crystal structures of GTFs to the peptide-pol II cocrystal structures. (3) Determination of Mediator structure. We propose to extend our structural analysis of the 7-subunit 220 kDa Mediator Head module, currently at 4.3 A, to higher resolution and to the complete 21-subunit 1 MDa Mediator complex.
The proposed research is relevant to both fundamental studies and to human health. The methods and results will contribute to the eventual understanding of all aspects of gene transcription. The elucidation of the transcription mechanism and its regulation will enable insights into cancer and other diseases, and will lead to novel therapeutics.
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|Liu, Xin; Bushnell, David A; Silva, Daniel-Adriano et al. (2011) Initiation complex structure and promoter proofreading. Science 333:633-7|
|Huang, Xuhui; Wang, Dong; Weiss, Dahlia R et al. (2010) RNA polymerase II trigger loop residues stabilize and position the incoming nucleotide triphosphate in transcription. Proc Natl Acad Sci U S A 107:15745-50|
|Liu, Xin; Bushnell, David A; Wang, Dong et al. (2010) Structure of an RNA polymerase II-TFIIB complex and the transcription initiation mechanism. Science 327:206-9|
|Wang, Dong; Zhu, Guangyu; Huang, Xuhui et al. (2010) X-ray structure and mechanism of RNA polymerase II stalled at an antineoplastic monofunctional platinum-DNA adduct. Proc Natl Acad Sci U S A 107:9584-9|
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|Kaplan, Craig D; Larsson, Karl-Magnus; Kornberg, Roger D (2008) The RNA polymerase II trigger loop functions in substrate selection and is directly targeted by alpha-amanitin. Mol Cell 30:547-56|
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