Transcription termination is a process whereby the elongation complex (EC) dissociates into RNA transcript, DNA template, and RNA polymerase (RNAP) in response to intrinsic signals or specific factors. Rho termination factor is essential in regulating gene expression in Enterobacter and is a target for specific antibiotics. Rho has been intensively studied in the last three decades, however, the actual termination process, i.e. the mechanism by which Rho disrupts the EC, remains unknown. Moreover, the identity of most Rho termination sites in vivo and the role of Rho in cell's adaptation to environmental changes remain unknown. The long-term objective of the proposed work is to provide a comprehensive physiological and mechanistic description of Rho-dependent termination in Escherichia coli and the mechanism of its regulation by particular host and phage proteins. Specifically we propose to: 1) Determine conformational changes in RNAP that accompany the termination process, and the role of certain RNAP domains in Rho termination. 2) Determine how E.coli S4 and phage l N proteins modify the EC rendering it resistant to Rho termination. 3) Determine the physiological role and mechanism of novel anti-Rho factors that we have identified in preliminary studies. 4) Establish the role of Rho in gene regulation on a genomic scale. The significance of proposed research for human health is several-fold. Complete structural understanding of termination/antitermination processes would allow for designing small molecule mimics and inhibitors that change the pattern of bacterial gene expression or interrupt transcription of essential genes prematurely, and thus serve as novel antimicrobials. Examples of antibiotics that specifically target Rho have been already described. Furthermore, better understanding the mechanisms of antitermination would suggest the optimal strategies for constructing bacterial strains that overproduce essential dietary supplements and other biologically active compounds. Finally, since eukaryotic RNAPs share basic sequence and structural homologies with bacterial RNAP, the fundamental mechanism of the EC stabilization and destabilization must be similar. Therefore, proposed experiments will also provide insight to the basic mechanisms of eukaryotic transcription termination. We will explore the mechanism of Rho termination and antitermination in E.coli. The significance of proposed research for human health is several-fold. Complete structural understanding of termination/antitermination processes would allow designing small molecule mimics and inhibitors of the termination process that change the pattern of bacterial gene expression or interrupt transcription of essential genes prematurely, and thus serve as novel antimicrobials. Furthermore, better understanding the mechanisms of antitermination would suggest optimal strategies for constructing bacterial strains that overproduce essential dietary supplements (vitamins, amino acids, etc.) and other important biologically active compounds. Finally, the proposed experiments will also provide insight to the basic mechanisms of eukaryotic transcription termination.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
3R01GM058750-13S1
Application #
8672454
Study Section
Program Officer
Sledjeski, Darren D
Project Start
2014-01-01
Project End
2015-12-31
Budget Start
2014-01-01
Budget End
2015-12-31
Support Year
13
Fiscal Year
2014
Total Cost
$169,500
Indirect Cost
$69,500
Name
New York University
Department
Biochemistry
Type
Schools of Medicine
DUNS #
121911077
City
New York
State
NY
Country
United States
Zip Code
10016
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