Our long-term objectives are to contribute to the understanding of the basic principles most important in gene regulation by learning how the AraC protein regulates transcription of the L-arabinose operon in Escherichia coli. Work will be done in three areas central to gene regulation. First, we will study the structure and function of the regulatory protein, AraC, itself. Genetic and physical approaches will be developed and applied for determining and studying domains within proteins, linker regions that connect domains, and identifying the amino acids that contact DNA. The domains of AraC and homologous domains from related proteins will be purified, and studied with the objective of determining their structures. The physical basis for DNA looping and unlooping phenomena in the ara system will be sought by studying the domain, flexible linker, and binding site preferences of the protein in the presence and absence of arabinose. Our second research area will be determining how regulatory proteins influence transcription by RNA polymerase. For this, we will use genetics, in vivo and in vitro transcription initiation measurements, and direct measurements of the interaction between AraC protein and RNA polymerase. We will test whether other members in the AraC family, which now numbers more than 20, use the same mechanisms for DNA binding and transcription activation. Our third research area will be on the integration of regulation of the ara genes into the overall physiology of cells. We will measure the in vivo regulatory properties of the five arabinose-regulated promoters in E. coli and relate the differences to the differences in the individual promoters and regulatory regions. We will also construct and test the behavior of new regulatory systems that use components from the arabinose operon and its relatives. The knowledge gained from our work should facilitate constructing systems with specified regulatory properties, and make it easier to manage the expression of natural gene systems in bacteria, plants, animals and viruses.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Method to Extend Research in Time (MERIT) Award (R37)
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Microbial Physiology and Genetics Subcommittee 2 (MBC)
Program Officer
Anderson, James J
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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