In this application we propose various complementary single molecule assays to investigate the mechanical translocation of individual E. coli RNA polymerase molecules during transcription elongation. Through these studies, we seek to determine the complex dynamics of RNA polymerase (RNAP) as it moves along the template and characterize the relationship between nucleotide addition and translocation as a first step to ultimately dissect the details of its mechanochemical cycle at high resolution. Specifically, we propose to: 1. Characterize pausing and arrest behavior during continued, uninterrupted elongation using an integrated optical trapping/flow control video microscope currently operating in our laboratory. 2. Characterize the mechanochemical cycle of the motor. To this end, we will obtain force-velocity curves for E. coli RNA polymerase under a variety of conditions. These curves will be analyzed using a molecular motor theory recently developed in our laboratory. 3. Develop a high resolution translocation assay to directly observe the dynamics of translocation of RNAP at single bp resolution. 4. Characterize the dynamics of translocation of RNAP against torsional stress by performing single molecule transcription assays with torsionally constrained DNA both in the presence and absence of E. coli gyrase. Through the single molecule studies described here, it will be possible to follow individual molecular events that would otherwise be missed in the ensemble average of bulk measurements. These events, involving dynamical changes in structure and function of the enzyme, lie at the heart of the process of regulation of gene expression. Finally, in the experiments described here, mechanical force will be used as a new controllable variable to characterize the process of mechanochemical transduction in this motor enzyme.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Method to Extend Research in Time (MERIT) Award (R37)
Project #
5R37GM032543-21
Application #
6519121
Study Section
Molecular and Cellular Biophysics Study Section (BBCA)
Program Officer
Lewis, Catherine D
Project Start
1983-07-01
Project End
2004-06-30
Budget Start
2002-07-01
Budget End
2003-06-30
Support Year
21
Fiscal Year
2002
Total Cost
$260,321
Indirect Cost
Name
University of California Berkeley
Department
Biochemistry
Type
Schools of Arts and Sciences
DUNS #
094878337
City
Berkeley
State
CA
Country
United States
Zip Code
94704
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