The goal of this research is to elucidate the molecular mechanisms involved in regulation of transcription, using as model systems two catabolite sensitive operons of E. coli. A variety of biophysical and biochemical methods will be applied to study interactions of the catabolite activator protein (CAP) and RNA polymerase with DNA fragments containing wild type or mutant promoter regions of the lactose and galactose operons. The techniques to be used include gel electrophoresis (for study of DNA-protein binding and dissociation as well as of transcription products), nuclease protection experiments which identify base sequences covered by DNA-bound proteins, centrifugation, electron microscopy, and optical methods such as fluorescence. Transcriptional initiation at the molecular level is more complex than originally thought. The gal promoter, for instance, is known to contain overlapping start sites for mRNA synthesis, one of which responds to CAP, the other not. Recent work on this project has shown that two CAP molecules are involved in stimulation of gal transcription. Likewise, while the lac operon shows a 1:1:1 stoichiometry for CAP:polymerase:promoter interactions, there is mounting evidence that CAP and RNA polymerase may interact in more than one way at the lac control region. The proposed research involves study of the biochemistry of these systems to characterize them and to clarify how these intricate regulatory mechanisms are modulated and fine-tuned in vivo. The techniques to be used (and developed as needed) will be applicable to study of specific, non-histone chromosomal proteins in mammalian organisms. The concepts derived from this work on a bacterial system will influence future research on control processes in eukaryotic cells. Ultimately, the philosophy underlying the project is that a detailed understanding of normal, regulatory processes is crucial to unraveling of the mysteries of uncontrolled, malignant cell growth and of other pathological conditions as well.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
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Biophysics and Biophysical Chemistry A Study Section (BBCA)
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Michigan State University
Schools of Osteopathy
East Lansing
United States
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Lorimer, D D; Cao, J L; Revzin, A (1990) Specific sequences downstream from -6 are not essential for proper and efficient in vitro utilization of the Escherichia coli lactose promoter. J Mol Biol 216:275-87
Ceglarek, J A; Revzin, A (1989) Studies of DNA-protein interactions by gel electrophoresis. Electrophoresis 10:360-5
Revzin, A (1989) Gel electrophoresis assays for DNA-protein interactions. Biotechniques 7:346-55
Shanblatt, S H; Revzin, A (1987) Interactions of the catabolite activator protein (CAP) at the galactose and lactose promoters of Escherichia coli probed by hydroxyl radical footprinting. The second CAP molecule which binds at gal and the one CAP at lac may act to stimulate transcription J Biol Chem 262:11422-7
Shanblatt, S H; Revzin, A (1986) Role of a second catabolite activator protein molecule in controlling initiation of transcription at the galactose operon of Escherichia coli. Biochemistry 25:5539-46
Shanblatt, S H; Revzin, A (1986) The binding of catabolite activator protein and RNA polymerase to the Escherichia coli galactose and lactose promoters probed by alkylation interference studies. J Biol Chem 261:10885-90
Lorimer, D D; Revzin, A (1986) Solutions of RNA polymerase plus linear wild type E. coli lac DNA fragments contain a mixture of stable P1 and P2 promoter complexes. Nucleic Acids Res 14:2921-38
Revzin, A; Ceglarek, J A; Garner, M M (1986) Comparison of nucleic acid-protein interactions in solution and in polyacrylamide gels. Anal Biochem 153:172-7