The prokaryotic transcript cleavage factor GreA and GreB are presumed to have two biologically important and evolutionarily conserved functions: the suppression of transcription arrest and the enhancement of transcription fidelity. These functions are accomplished by the ability of Gre factors to induce the cleavage of the nascent RNA in ternary complexes of RNA polymerase. The broad goal of this project is to understand the molecular mechanism of action and the structure-functional relationships of GreA and GreB in Escherichia coli. For this purpose, three types of experiments will be conducted. First, to identify functionally important localities of Gre factors, the amino acid residues of Gre A and Gre B that, according to their established 3-D structure, are located on the protein surface will be mutagenized. The mutant proteins will be then characterized biochemically using specific in vitro transcription assays and structurally by X-ray analyses. The second type of experiments are aimed at detailed studies of the basic """"""""patches"""""""" of Gre molecules formed by positively charged surface-exposed residues. To this end, a series of Gre mutants will be constructed that carry basic patches of various sizes and the resulting mutant factors will be analyzed functionally an biochemically. A model that implicates the basic residues of the patches in activation of the intrinsic nucleolytic site in RNA polymerase will be tested. Finally, the interactions of Gre A and Gre B with RNA polymerase will be studied using protein-protein photochemical crosslinking. The Cys residues will be introduced into Gre proteins by site-directed mutagenesis of selected surface-exposed residues followed by their derivatization with thiol-specific photoactive bifunctional reagents. The resulting modified Gre proteins will be used to probe the interactions with RNA polymerase at different stages of transcription elongation.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM054098-02
Application #
2415358
Study Section
Microbial Physiology and Genetics Subcommittee 2 (MBC)
Project Start
1996-05-01
Project End
2001-04-30
Budget Start
1997-05-01
Budget End
1998-04-30
Support Year
2
Fiscal Year
1997
Total Cost
Indirect Cost
Name
Suny Downstate Medical Center
Department
Microbiology/Immun/Virology
Type
Schools of Medicine
DUNS #
068552207
City
Brooklyn
State
NY
Country
United States
Zip Code
11203
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