Abstract

The cAMP receptor protein (CRP) plays a key role in transcription of catabolite repressible operons as a positive regulatory factor via cAMP dependent DNA (promoter) binding. The proposed research involves an investigation of the role of CRP structure and function for cAMP and DNA binding. CRP has a domain structure in which the amino proximal region is involved in binding cAMP and subunit-subunit interaction while the carboxyl proximal region is involved in DNA binding. Polypeptide fragments and cores have been generated by limited proteolysis of CRP; these will be studied with regard to reconstitution of CRP activity, DNA binding and possible regulatory properties for in vitro lac and gal operon expression. Modification of CRP at the carboxyl terminus by carboxypeptidase will be carried out to determine the role of this region in DNA binding and CRP conformation. Chemical modification studies will be continued to determine effects on CRP structure and function. A temperature sensitive CRP mutant will be purified and characterized in terms of the ts lesion; revertants will be selected for study which show properties different from that of wild type CRP. Using hybridomas monoclonal antibodies will be prepared against CRP fragments. These monospecific antibodies will be used as probes of CRP conformation and function.

  Funding Agency

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM022619-12
Application #
3271227
Study Section
Microbial Physiology and Genetics Subcommittee 2 (MBC)
Project Start
1976-04-01
Project End
1990-08-31
Budget Start
1987-09-01
Budget End
1988-08-31
Support Year
12
Fiscal Year
1987
Total Cost
Indirect Cost

  Institution

Name
Hunter College
Department
Type
Schools of Arts and Sciences
DUNS #
City
New York
State
NY
Country
United States
Zip Code
10065

  Publications

Sharif, K A; Luo, J; Krakow, J S (1996) Epitope mapping of monoclonal antibodies directed against subunits of RNA polymerase of Escherichia coli. Methods Enzymol 274:503-13
Luo, J; Sharif, K A; Jin, R et al. (1996) Molecular anatomy of the beta' subunit of the E. coli RNA polymerase: identification of regions involved in polymerase assembly. Genes Cells 1:819-27
Jin, R; Sharif, K A; Krakow, J S (1995) Evidence for contact between the cyclic AMP receptor protein and the delta 70 subunit of Escherichia coli RNA polymerase. J Biol Chem 270:19213-6
Sharif, K A; Fujita, N; Jin, R et al. (1994) Epitope mapping and functional characterization of monoclonal antibodies specific for the alpha subunit of Escherichia coli RNA polymerase. J Biol Chem 269:23655-60
Luo, J; Krakow, J S (1992) Characterization and epitope mapping of monoclonal antibodies directed against the beta' subunit of the Escherichia coli RNA polymerase. J Biol Chem 267:18175-81
Yang, Z H; Bobin, S; Krakow, J S (1991) Characterization of the CRPCY core formed after treatment with carboxypeptidase Y. Nucleic Acids Res 19:4253-7
Ren, Y L; Garges, S; Adhya, S et al. (1990) Characterization of the binding of cAMP and cGMP to the CRP*598 mutant of the E. coli cAMP receptor protein. Nucleic Acids Res 18:5127-32
Ebright, R H; Gunasekera, A; Zhang, X P et al. (1990) Lysine 188 of the catabolite gene activator protein (CAP) plays no role in specificity at base pair 7 of the DNA half site. Nucleic Acids Res 18:1457-64
Li, X M; Krakow, J S (1988) Monoclonal antibodies that inhibit activation of transcription by the Escherichia coli cyclic AMP receptor protein. J Biol Chem 263:3448-53
Ren, Y L; Garges, S; Adhya, S et al. (1988) Cooperative DNA binding of heterologous proteins: evidence for contact between the cyclic AMP receptor protein and RNA polymerase. Proc Natl Acad Sci U S A 85:4138-42

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