This grant supported the development of a yeast RNA polymerase II transcription system, leading over the past 20 years to the following: isolation of the general transcription factors TFIIB, -D, -E, F, and -H, and the reconstitution of transcription with pure proteins;isolation of the genes for the 16 subunits of the general factors, revealing a remarkable degree of conservation from yeast to humans;discovery of the multiple roles of TFIIH in transcription and DNA repair;discovery of the Mediator of transcriptional regulation;and provision of RNA polymerase II and general transcription factors for X-ray and electron crystal structure determination. The methods developed for these purposes were exploited in the isolation of chromatin remodeling complexes and, most recently, the isolation of genes in their native states. Characterization of the isolated genes revealed the disruption of promoter nucleosomes upon transcriptional activation in vivo. We now wish to reproduce the entire gene activation process in vitro, beginning with a gene in its repressed state, and finishing with transcription. We tried unsuccessfully to establish a gene activation system for the past 20 years. Positive results from a recent breakthrough in the work form a major part of this proposal. We wish to isolate the factors involved and investigate the mechanism of promoter chromatin remodeling. The block to remodeling imposed by heterochromatin will be both amenable to study in the purified system and a source of information about the remodeling mechanism. In parallel with the studies of chromatin remodeling, we will pursue the transcription of naked DNA templates with RNA polymerase II and general transcription factors. Our work of the past several years has overcome longstanding technical limitations and yielded essentially homogeneous, fully assembled pre-initiation complexes. We will introduce purified Mediator and activator proteins to bring about efficient template utilization. Finally, we wish to combine both promoter remodeling and transcription systems to reconstitute the entire gene activation process.
Specific aims for the next project period are as follows: 1. Resolution and reconstitution of the newly established PHO5 promoter chromatin remodeling system. 2. Identification of the block(s) to promoter chromatin remodeling of the PHO5 promoter in heterochromatin. 3. Efficient initiation of RNA polymerase II transcription by fully assembled pre-initiation complexes. 4. Coupling of promoter chromatin remodeling and transcription initiation in a complete gene activation system.

Public Health Relevance

The significance of the proposed research may be summarized as follows: it will provide the information needed to fully understand the fundamental mechanism of transcription;it will establish a 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 General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM036659-25
Application #
7992362
Study Section
Molecular Genetics A Study Section (MGA)
Program Officer
Tompkins, Laurie
Project Start
1986-07-01
Project End
2012-11-30
Budget Start
2010-12-01
Budget End
2011-11-30
Support Year
25
Fiscal Year
2011
Total Cost
$599,997
Indirect Cost
Name
Stanford University
Department
Biology
Type
Schools of Medicine
DUNS #
009214214
City
Stanford
State
CA
Country
United States
Zip Code
94305
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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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