Abstract

Transcription is the major control point of gene expression and RNA polymerase (RNAP) is the central enzyme of transcription. Our long term goal is to understand the mechanism of transcription and its regulation. Determining three-dimensional structures of RNAP and its complexes with DNA, RNA, and regulatory, is an essential step. This is best accomplished with highly characterized prokaryotic RNAPs, especially because of the high degree of conservation of RNAP structure and function from bacteria to man. To this end, we recently purified, crystallized, and determined the 3.3 angstrom units-resolution X-ray crystal structure of a prokaryotic RNAP, core RNAP from the thermophilic eubacteria Thermus aquaticus (taq). This represents a major breakthrough in our work. Here we propose further structural studies, all aimed towards adding to our understanding of the enzyme's function and its regulation. Specifically, we propose to: 1. Complete the refinement of the taq core RNAP model 2. Determine the 3.0 angstrom units crystal structure of taq core RNAP complexed with antibiotic inhibitors. 3. Determine the structure of taq core RNAP complexed with antibiotic inhibitors. 4. Determine the structure of taq RNAP holoenzyme. 5. Determine the structure of a binary complex between taq core RNAP and template DNA. 6. Determine the structure of an active ternary elongation complex between taq core RNAP, DNA template, and RNA transcript. These studies will have a wide-ranging impact on studies of transciption.

  Funding Agency

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM061898-04
Application #
6657438
Study Section
Molecular and Cellular Biophysics Study Section (BBCA)
Program Officer
Lewis, Catherine D
Project Start
2000-09-01
Project End
2004-08-31
Budget Start
2003-09-01
Budget End
2004-08-31
Support Year
4
Fiscal Year
2003
Total Cost
$334,000
Indirect Cost

  Institution

Name
Rockefeller University
Department
Physiology
Type
Other Domestic Higher Education
DUNS #
071037113
City
New York
State
NY
Country
United States
Zip Code
10065

  Publications

Berdygulova, Zhanna; Westblade, Lars F; Florens, Laurence et al. (2011) Temporal regulation of gene expression of the Thermus thermophilus bacteriophage P23-45. J Mol Biol 405:125-42
Lane, William J; Darst, Seth A (2010) Molecular evolution of multisubunit RNA polymerases: structural analysis. J Mol Biol 395:686-704
Lane, William J; Darst, Seth A (2010) Molecular evolution of multisubunit RNA polymerases: sequence analysis. J Mol Biol 395:671-85
Opalka, Natacha; Brown, Jesse; Lane, William J et al. (2010) Complete structural model of Escherichia coli RNA polymerase from a hybrid approach. PLoS Biol 8:
Westblade, Lars F; Minakhin, Leonid; Kuznedelov, Konstantin et al. (2008) Rapid isolation and identification of bacteriophage T4-encoded modifications of Escherichia coli RNA polymerase: a generic method to study bacteriophage/host interactions. J Proteome Res 7:1244-50
Feklistov, Andrey; Mekler, Vladimir; Jiang, Qiaorong et al. (2008) Rifamycins do not function by allosteric modulation of binding of Mg2+ to the RNA polymerase active center. Proc Natl Acad Sci U S A 105:14820-5
Savalia, Dhruti; Westblade, Lars F; Goel, Manisha et al. (2008) Genomic and proteomic analysis of phiEco32, a novel Escherichia coli bacteriophage. J Mol Biol 377:774-89
Murakami, Katsuhiko S; Davydova, Elena K; Rothman-Denes, Lucia B (2008) X-ray crystal structure of the polymerase domain of the bacteriophage N4 virion RNA polymerase. Proc Natl Acad Sci U S A 105:5046-51
Lee, David J; Busby, Stephen J W; Westblade, Lars F et al. (2008) Affinity isolation and I-DIRT mass spectrometric analysis of the Escherichia coli O157:H7 Sakai RNA polymerase complex. J Bacteriol 190:1284-9
Bai, Yu; Markham, Kelly; Chen, Fusheng et al. (2008) The in vivo brain interactome of the amyloid precursor protein. Mol Cell Proteomics 7:15-34

Showing the most recent 10 out of 26 publications