This grant supports our studies of yeast RNA polymerase II (pol II) transcription. The long range goal of the work has been and remains to discover the mechanism of transcription and its regulation. The chief obstacle to progress has long been the requirement for abundant, monodisperse, pure GTFs and Mediator for mechanistic studies. Our work over the past twenty years has removed this obstacle, with the most intractable components, TFIIH and Mediator, having been obtained in suitable form during the previous project period. Three advances form the basis for the present proposal: our isolation in stable, homogeneous, fully functional form of a complete PIC (giant complex of pol II, all GTFs, and promoter DNA);our success, after decades of failed attempts, in establishing a gene activation system, starting from a gene isolated as chromatin in the repressed state, and giving rise to a high level of transcription;and developments both in mass spectrometry, giving 100-fold greater sensitivity, and in electron microscopy, stemming from our first synthesis of molecularly defined gold nanoparticles.
Specific aims for the next project period are three-fold: to fractionate our gene activation system to elucidate the remodeling of chromatin structure for transcription, with the use of electron microscopy and mass spectrometry;and to investigate transcription initiation in a complete PIC by "walking" the early transcript and by single molecule analysis.

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 chromosomal DNA transactions. The elucidation of the gene activation process in particular will enable insights int 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 #
5R01GM036659-28
Application #
8636475
Study Section
Molecular Genetics A Study Section (MGA)
Program Officer
Sledjeski, Darren D
Project Start
1986-07-01
Project End
2016-11-30
Budget Start
2013-12-01
Budget End
2014-11-30
Support Year
28
Fiscal Year
2014
Total Cost
$497,313
Indirect Cost
$180,553
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
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
Lorch, Yahli; Maier-Davis, Barbara; Kornberg, Roger D (2014) Role of DNA sequence in chromatin remodeling and the formation of nucleosome-free regions. Genes Dev 28:2492-7
Azubel, Maia; Koivisto, Jaakko; Malola, Sami et al. (2014) Nanoparticle imaging. Electron microscopy of gold nanoparticles at atomic resolution. Science 345:909-12
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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