The long-term objective of this project is to understand as fully and as deeply as possible regulation of the L-arabinose operon in the bacterium Escherichia coli. In the short term, questions as outlined below will be addressed. In the longer term, as in all research, the objective is to understand important regulatory phenomena so that we may use this knowledge in health or medical issues and also design and build other systems using the same principles. I am focusing on the arabinose operon and its regulatory protein AraC because it is one of a small number of intensively studied model systems. It displays a variety of transcriptional regulatory phenomena while at the same time is sufficiently simple as to allow genetic, physiological, in vitro, biochemical, and biophysical approaches. Experiments will be done in two general areas. The main body of work will be the determination by NMR of the structure of the DNA binding domain of AraC and related proteins or protein complexes. These include: the DNA binding domain with and without the regulatory N-terminal arm bound to it, the structures when bound to a tight-binding DNA site and a weak-binding site, and the structures of a number of mutants in the arm and in the DNA binding domain that strengthen or weaken binding of the arm. We will also investigate a number of mechanistic questions. These will be centered around the interaction between the regulatory arm and the AraC DNA binding domain. The affinity of the arm for the domain will be measured for wild type regulatory arm and domain and for a number of mutants. We will also look for an additional interaction between the dimerization domain that functions only in the repression state. We will determine the mechanisms by which TrpR and TetR regulate their DNA binding affinity in response to regulating small molecule ligands. Regulatory arms bind to other proteins. What happens when an arm does not bind other proteins or domains? Does it repel other proteins? We will examine this question and the possibility of developing a regulatory mechanism based upon it.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
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Prokaryotic Cell and Molecular Biology Study Section (PCMB)
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Tompkins, Laurie
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Johns Hopkins University
Schools of Arts and Sciences
United States
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Seedorff, Jennifer; Schleif, Robert (2011) Active role of the interdomain linker of AraC. J Bacteriol 193:5737-46
Berrondo, Monica; Gray, Jeffrey J; Schleif, Robert (2010) Computational predictions of the mutant behavior of AraC. J Mol Biol 398:462-70
Seedorff, Jennifer E; Rodgers, Michael E; Schleif, Robert (2009) Opposite allosteric mechanisms in TetR and CAP. Protein Sci 18:775-81
Rodgers, Michael E; Schleif, Robert (2009) Solution structure of the DNA binding domain of AraC protein. Proteins 77:202-8
Rodgers, Michael E; Holder, Nakisha D; Dirla, Stephanie et al. (2009) Functional modes of the regulatory arm of AraC. Proteins 74:81-91
Dirla, Stephanie; Chien, John Yeh-Heng; Schleif, Robert (2009) Constitutive mutations in the Escherichia coli AraC protein. J Bacteriol 191:2668-74
Frato, Katherine E; Schleif, Robert F (2009) A DNA-assisted binding assay for weak protein-protein interactions. J Mol Biol 394:805-14
Hargreaves, Victoria V; Schleif, Robert F (2008) The salt dependence of the interferon regulatory factor 1 DNA binding domain binding to DNA reveals ions are localized around protein and DNA. Biochemistry 47:4119-28