Abstract

This proposal is designed to determine the mechanism of elongation by human RNA polymerase II (RNAP II) and to elucidate combinatorial control by elongation factors. The Burton laboratory has pioneered and verified rapid chemical quench-flow kinetic methods to analyze RNAP II functional dynamics during elongation in real time (millisecond phase), using combinations of potent regulators.
The Specific Aims are to: 1) characterize the RNAP II elongation control system, develop and refine a kinetic mechanism for elongation by human RNAP II, and challenge the NTP driven translocation model for RNAP II elongation; 2) determine the roles of viral hepatitis delta antigen (HDAg), human Transcription Factor IIF (TFIIF), and human TFIIS in regulation of RNAP II elongation, and use these factors as probes of the RNAP II mechanism; 3) identify mechanisms for fidelity and efficiency of RNAP II elongation; and 4) initiate rapid quench-flow studies of yeast RNAP II elongation to exploit the yeast RNAP II elongation control proteome. Kinetic analysis of human RNAP II gives novel insight into the mechanism and regulation of translocation, NTP loading, pyrophosphate release and the efficiency and fidelity of NMP incorporation.

  Funding Agency

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
2R01GM057461-05A1
Application #
6865108
Study Section
Biochemistry Study Section (BIO)
Program Officer
Tompkins, Laurie
Project Start
2000-03-01
Project End
2008-08-31
Budget Start
2004-09-15
Budget End
2005-08-31
Support Year
5
Fiscal Year
2004
Total Cost
$299,046
Indirect Cost

  Institution

Name
Michigan State University
Department
Biochemistry
Type
Schools of Arts and Sciences
DUNS #
193247145
City
East Lansing
State
MI
Country
United States
Zip Code
48824

  Publications

Domecq, Céline; Kireeva, Maria; Archambault, Jacques et al. (2010) Site-directed mutagenesis, purification and assay of Saccharomyces cerevisiae RNA polymerase II. Protein Expr Purif 69:83-90
Feig, Michael; Burton, Zachary F (2010) RNA polymerase II flexibility during translocation from normal mode analysis. Proteins 78:434-46
Seibold, Steve A; Singh, Badri Nath; Zhang, Chunfen et al. (2010) Conformational coupling, bridge helix dynamics and active site dehydration in catalysis by RNA polymerase. Biochim Biophys Acta 1799:575-87
Kireeva, Maria; Nedialkov, Yuri A; Gong, Xue Qian et al. (2009) Millisecond phase kinetic analysis of elongation catalyzed by human, yeast, and Escherichia coli RNA polymerase. Methods 48:333-45
Thompson, Nancy E; Glaser, Bryan T; Foley, Katherine M et al. (2009) Minimal promoter systems reveal the importance of conserved residues in the B-finger of human transcription factor IIB. J Biol Chem 284:24754-66
Xiong, Yalin; Burton, Zachary F (2007) A tunable ratchet driving human RNA polymerase II translocation adjusted by accurately templated nucleoside triphosphates loaded at downstream sites and by elongation factors. J Biol Chem 282:36582-92