The promoter and regulatory region of the ilvlH operon of Escherichia coli K-12 has three interesting features: dependence of expression upon relatively distant upstream sequences, high- affinity binding to a rare E. coli protein, and anomalous migration during electrophoresis. Preliminary experiments indicate that some unusual structural feature underlies the phenomenon of anomalous migration. The goals of this research are to investigate these features and possible relationships between them. At present, there are few examples of DNA regions having an unusual structural feature of the type manifested by the ilvlH regulatory region and almost nothing is known about the underlying basis for such unusual structural features and whether gene expression is influenced by them. Protection and binding interference experiments will be carried out to define sites at which binding occurs. In addition, the size and subunit composition of the binding protein will be determined and the binding constant and stoichiometry of binding will be measured. Mutations will be made within the ilvlH promoter and regulatory region using site-directed procedures and the resulting mutations will be analyzed for their effects upon in vivo expression of the operon, in vitro transcription, binding, and anomalous migration. In addition, mutations within the locus that codes for the binding protein will be isolated to determine the effect that null alleles have upon expression of the ilvlH operon. Finally, experiments will be carried out to investigate the basis for anomalous migration. The overall goals of this research are to understand how DNA structure affects gene expression. Advances in medicine depend upon such basic information.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
1R01GM039496-01
Application #
3296521
Study Section
Microbial Physiology and Genetics Subcommittee 2 (MBC)
Project Start
1988-02-01
Project End
1993-01-31
Budget Start
1988-02-01
Budget End
1989-01-31
Support Year
1
Fiscal Year
1988
Total Cost
Indirect Cost
Name
Cornell University
Department
Type
Schools of Arts and Sciences
DUNS #
City
Ithaca
State
NY
Country
United States
Zip Code
14850
Chen, Shaolin; Iannolo, Maria; Calvo, Joseph M (2005) Cooperative binding of the leucine-responsive regulatory protein (Lrp) to DNA. J Mol Biol 345:251-64
Chen, Shaolin; Calvo, Joseph M (2002) Leucine-induced dissociation of Escherichia coli Lrp hexadecamers to octamers. J Mol Biol 318:1031-42
Jafri, Samina; Chen, Shaolin; Calvo, Joseph M (2002) ilvIH operon expression in Escherichia coli requires Lrp binding to two distinct regions of DNA. J Bacteriol 184:5293-300
Chen, S; Rosner, M H; Calvo, J M (2001) Leucine-regulated self-association of leucine-responsive regulatory protein (Lrp) from Escherichia coli. J Mol Biol 312:625-35
Chen, S; Hao, Z; Bieniek, E et al. (2001) Modulation of Lrp action in Escherichia coli by leucine: effects on non-specific binding of Lrp to DNA. J Mol Biol 314:1067-75
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Cui, Y; Midkiff, M A; Wang, Q et al. (1996) The leucine-responsive regulatory protein (Lrp) from Escherichia coli. Stoichiometry and minimal requirements for binding to DNA. J Biol Chem 271:6611-7
Cui, Y; Wang, Q; Stormo, G D et al. (1995) A consensus sequence for binding of Lrp to DNA. J Bacteriol 177:4872-80
Calvo, J M; Matthews, R G (1994) The leucine-responsive regulatory protein, a global regulator of metabolism in Escherichia coli. Microbiol Rev 58:466-90
Wang, Q; Calvo, J M (1993) Lrp, a major regulatory protein in Escherichia coli, bends DNA and can organize the assembly of a higher-order nucleoprotein structure. EMBO J 12:2495-501

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