This grant supported the development of two-dimensional (2D) protein crystallography, which led, over the course of 25 years, to electron and X-ray crystal structures of RNA polymerase II (pol II), alone and in the act of transcription. These structures were extended, during the previous project period (2002-2007), to higher complexes with general transcription factors, illuminating the mechanisms of transcription start site selection, promoter melting, abortive initiation, and promoter escape. We now propose the culmination of our efforts, the structure determination of the entire transcription machinery. Biochemical studies of the general transcription factors and of the Mediator of transcriptional regulation during the previous project period have removed the major obstacles to the proposed research. Preliminary crystallographic work has shown the feasibility of accomplishing our goal during the next project period. In parallel with the application of 2D crystallography and other methods to the transcription problem, we have pursued the development of the methodology itself. This work was inspired by an idea for routine high resolution structure determination of large complexes by electron microscopy and single particle analysis. We have proposed to rigidly attach multiple heavy atom clusters to specific sites on the complexes before imaging, and then align the images based on the projected positions of the clusters. This heavy atom electron microscopy, or "HevEM," approach requires perfectly uniform clusters about a nanometer in diameter, as well as chemistries for their conjugation with biological molecules. During the past decade, we have devised procedures for meeting these requirements, and are poised for the first trial of HevEM, as well as its application to the giant transcription protein assemblies of interest.
Specific aims for the next project period are as follows: 1. Structure determination of general transcription factor H. 2. Structure determination of pol II - general transcription factor complexes. 3. Structure determination of Mediator and of Mediator - pol II complexes. 4. Development of the HevEM approach.

Public Health Relevance

The significance of the proposed research may be summarized as follows: it will provide the structural information needed to fully understand the fundamental mechanism of transcription;it will establish a structural basis for studies of transcriptional regulation;and it will facilitate the design of new therapies for diseases of aberrant gene regulation in the future.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI021144-28
Application #
8416447
Study Section
Macromolecular Structure and Function B Study Section (MSFB)
Program Officer
Leitner, Wolfgang W
Project Start
1987-05-01
Project End
2013-12-31
Budget Start
2013-01-01
Budget End
2013-12-31
Support Year
28
Fiscal Year
2013
Total Cost
$587,618
Indirect Cost
$220,357
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
Eagen, Kyle P; Hartl, Tom A; Kornberg, Roger D (2015) Stable Chromosome Condensation Revealed by Chromosome Conformation Capture. Cell 163:934-46
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
Lorch, Yahli; Kornberg, Roger D (2015) Chromatin-remodeling and the initiation of transcription. Q Rev Biophys 48:465-70
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
Robinson, Philip J; Trnka, Michael J; Pellarin, Riccardo et al. (2015) Molecular architecture of the yeast Mediator complex. Elife 4:
Schweikhard, Volker; Meng, Cong; Murakami, Kenji et al. (2014) Transcription factors TFIIF and TFIIS promote transcript elongation by RNA polymerase II by synergistic and independent mechanisms. Proc Natl Acad Sci U S A 111:6642-7

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