This project is designed to investigate important questions about the mechanism and regulation of transcription initiation in Escherichia coli. The experiments are designed to provide a fundamental biochemical understanding of several complex phenomena involved in the recognition of E. coli promoters by RNA polymerase. A series of studies will characterize the """"""""closed complex"""""""", an important reaction intermediate in the initiation process. Experiments performed in vitro will also characterize the properties of a mutant sigma subunit that displays altered promoter recognition in vivo. The effects of DNA supercoiling on promoter recognition will be explored using a newly-developed assay for topoisomer selectivity. This novel approach will be extended in a study of positive regulation by cap*cAMP at the lactose and galactose operon promoters. The modulation of promoter selectivity by the galactose repressor at the gal promoters will also be characterized. The structural determinants of RNA-RNA pairing will also be determined in an example of antisense RNA gene regulation. In this case the regulation occurs primarily at the translation initiation steps, however, the principles for RNA-RNA pairing that emerge will be applicable to many other steps in gene expression and its regulation. The answers to the questions we have posed will contribute to the biochemical description of the control of gene expression.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM030375-13
Application #
3278123
Study Section
Bacteriology and Mycology Subcommittee 2 (BM)
Project Start
1981-07-01
Project End
1995-06-30
Budget Start
1993-07-01
Budget End
1994-06-30
Support Year
13
Fiscal Year
1993
Total Cost
Indirect Cost
Name
Carnegie-Mellon University
Department
Type
Schools of Arts and Sciences
DUNS #
052184116
City
Pittsburgh
State
PA
Country
United States
Zip Code
15213
Li, X Y; McClure, W R (1998) Stimulation of open complex formation by nicks and apurinic sites suggests a role for nucleation of DNA melting in Escherichia coli promoter function. J Biol Chem 273:23558-66
Li, X Y; McClure, W R (1998) Characterization of the closed complex intermediate formed during transcription initiation by Escherichia coli RNA polymerase. J Biol Chem 273:23549-57
Schaefer, K L; McClure, W R (1997) Antisense RNA control of gene expression in bacteriophage P22. II. Kinetic mechanism and cation specificity of the pairing reaction. RNA 3:157-74
Schaefer, K L; McClure, W R (1997) Antisense RNA control of gene expression in bacteriophage P22. I. Structures of sar RNA and its target, ant mRNA. RNA 3:141-56
Li, M; McClure, W R; Susskind, M M (1997) Changing the mechanism of transcriptional activation by phage lambda repressor. Proc Natl Acad Sci U S A 94:3691-6
Yee, Y C; Kisslinger, B; Yu, V L et al. (1996) A mechanism of rifamycin inhibition and resistance in Pseudomonas aeruginosa. J Antimicrob Chemother 38:133-7
Su, T T; McClure, W R (1994) Selective binding of Escherichia coli RNA polymerase to topoisomers of minicircles carrying the TAC16 and TAC17 promoters. J Biol Chem 269:13511-21
Goodrich, J A; McClure, W R (1992) Regulation of open complex formation at the Escherichia coli galactose operon promoters. Simultaneous interaction of RNA polymerase, gal repressor and CAP/cAMP. J Mol Biol 224:15-29
Goodrich, J A; McClure, W R (1991) Competing promoters in prokaryotic transcription. Trends Biochem Sci 16:394-7
Goodrich, J A; Schwartz, M L; McClure, W R (1990) Searching for and predicting the activity of sites for DNA binding proteins: compilation and analysis of the binding sites for Escherichia coli integration host factor (IHF). Nucleic Acids Res 18:4993-5000

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