Abstract

The objective of this work is to elucidate the detailed mechanism of control of transcription initiation at the E. coli lac promoter, with particular attention to the cAMP binding gene activating protein CAP.
Specific aims i nclude determination of the relationship between DNA sequence and the properties of the CAP- induced DNA bend, along with investigation of the relationship between the magnitude of the induced bend and the intrinsic bending in the naked DNA. We will measure the size of the protein-induced bend, and determine the contribution of writhe to the bend. Experiments that focus on RNA polymerase and transcription will include tracking the CAP-induced bend through the closed, open and initiated complexes. The position, binding stability and reaction kinetics of CAP and polymerase will be examined for promoter mutants with altered CAP sites and bending properties, and with altered phasing between the CAP and polymerase binding sites. Transcription activation in vitro and in vivo will also be studied from these mutants. The interaction of RNA polymerase in the presence of lac repressor will also be examined, and exploratory work on the interaction of a murine homeo domain protein with DNA will be pursued. Methods used are largely a combination of gel electrophoretic separation of protein-DNA complexes, together with chemical and enzymatic characterization of the interactions that stabilize binding.

  Funding Agency

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM034205-06
Application #
3284781
Study Section
Microbial Physiology and Genetics Subcommittee 2 (MBC)
Project Start
1984-12-01
Project End
1992-11-30
Budget Start
1989-12-01
Budget End
1990-11-30
Support Year
6
Fiscal Year
1990
Total Cost
Indirect Cost

  Institution

Name
Yale University
Department
Type
Schools of Arts and Sciences
DUNS #
082359691
City
New Haven
State
CT
Country
United States
Zip Code
06520

  Publications

Gartenberg, M R; Crothers, D M (1991) Synthetic DNA bending sequences increase the rate of in vitro transcription initiation at the Escherichia coli lac promoter. J Mol Biol 219:217-30
Zinkel, S S; Crothers, D M (1991) Catabolite activator protein-induced DNA bending in transcription initiation. J Mol Biol 219:201-15
Gartenberg, M R; Ampe, C; Steitz, T A et al. (1990) Molecular characterization of the GCN4-DNA complex. Proc Natl Acad Sci U S A 87:6034-8
Straney, D C; Straney, S B; Crothers, D M (1989) Synergy between Escherichia coli CAP protein and RNA polymerase in the lac promoter open complex. J Mol Biol 206:41-57
Brown, A M; Crothers, D M (1989) Modulation of the stability of a gene-regulatory protein dimer by DNA and cAMP. Proc Natl Acad Sci U S A 86:7387-91
Ghosaini, L R; Brown, A M; Sturtevant, J M (1988) Scanning calorimetric study of the thermal unfolding of catabolite activator protein from Escherichia coli in the absence and presence of cyclic mononucleotides. Biochemistry 27:5257-61
Straney, S B; Crothers, D M (1987) Lac repressor is a transient gene-activating protein. Cell 51:699-707
Straney, D C; Crothers, D M (1987) A stressed intermediate in the formation of stably initiated RNA chains at the Escherichia coli lac UV5 promoter. J Mol Biol 193:267-78
Straney, D C; Crothers, D M (1987) Effect of drug-DNA interactions upon transcription initiation at the lac promoter. Biochemistry 26:1987-95
Straney, D C; Crothers, D M (1987) Comparison of the open complexes formed by RNA polymerase at the Escherichia coli lac UV5 promoter. J Mol Biol 193:279-92

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