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.

Public Health Relevance

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.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM049985-21
Application #
8649043
Study Section
Macromolecular Structure and Function C Study Section (MSFC)
Program Officer
Flicker, Paula F
Project Start
1993-08-01
Project End
2017-01-31
Budget Start
2014-02-01
Budget End
2015-01-31
Support Year
21
Fiscal Year
2014
Total Cost
$504,419
Indirect Cost
$183,133
Name
Stanford University
Department
Biology
Type
Schools of Medicine
DUNS #
009214214
City
Stanford
State
CA
Country
United States
Zip Code
94305
Robinson, Philip J; Trnka, Michael J; Bushnell, David A et al. (2016) Structure of a Complete Mediator-RNA Polymerase II Pre-Initiation Complex. Cell 166:1411-1422.e16
Lu, Jonathan; Trnka, Michael J; Roh, Soung-Hun et al. (2015) Improved Peak Detection and Deconvolution of Native Electrospray Mass Spectra from Large Protein Complexes. J Am Soc Mass Spectrom 26:2141-51
Murakami, Kenji; Mattei, Pierre-Jean; Davis, Ralph E et al. (2015) Uncoupling Promoter Opening from Start-Site Scanning. Mol Cell 59:133-8
Guan, Shenheng; Trnka, Michael J; Bushnell, David A et al. (2015) Deconvolution method for specific and nonspecific binding of ligand to multiprotein complex by native mass spectrometry. Anal Chem 87:8541-6
Murakami, Kenji; Tsai, Kuang-Lei; Kalisman, Nir et al. (2015) Structure of an RNA polymerase II preinitiation complex. Proc Natl Acad Sci U S A 112:13543-8
Fazal, Furqan M; Meng, Cong A; Murakami, Kenji et al. (2015) Real-time observation of the initiation of RNA polymerase II transcription. Nature 525:274-7
Azubel, Maia; Koivisto, Jaakko; Malola, Sami et al. (2014) Nanoparticle imaging. Electron microscopy of gold nanoparticles at atomic resolution. Science 345:909-12
Liu, Xin; Bushnell, David A; Kornberg, Roger D (2013) RNA polymerase II transcription: structure and mechanism. Biochim Biophys Acta 1829:2-8
Robinson, Philip J J; Bushnell, David A; Trnka, Michael J et al. (2012) Structure of the mediator head module bound to the carboxy-terminal domain of RNA polymerase II. Proc Natl Acad Sci U S A 109:17931-5
Liu, Xin; Bushnell, David A; Silva, Daniel-Adriano et al. (2011) Initiation complex structure and promoter proofreading. Science 333:633-7

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