This project will investigate by a variety of in vivo and in vitro techniques the mechanism of action of araC protein in positively and negatively regulating initiation of transcription on the three sets of genes necessary for the uptake and catabolism of L-arabinose in Escherichia coli. Ultimately we wish to have a detailed model of the structure and function of this protein. The DNA binding properties and effects on transcription initiation by araC and cyclic AMP receptor proteins will be carefully investigated in vitro. Questions to be answered are: which bases and phosphates are necessary for protein binding and function; do the proteins accelerate the binding or isomerization rate of RNA polymerase; do the proteins affect one another in binding or dissociation rates or do they alter one another's conformations as is suggested by current experiments. Direct measurements and indirect physiological experiments will determine in vivo DNA binding properties of araC protein. DNA binding in vitro will be assayed by footprinting, transcription, and a new assay for araC protein based on araC-generated reduction in the electrophoretic mobility of a restriction fragment. Many mutations and deletions in the protein binding sites will be constructed and used in the above studies. Spectroscopic, tritium exchange, protease sensitivity, or antibody experiments will be used to assay for a conformational change in araC after it has bound to DNA. Classical experiments indicate that pBAD is repressed from a site upstream of the induction sites, but current data indicate the situation is more complex. In vivo and in vitro experiments will investigate this as well as related questions on the mechanism of regulation of the promoter for synthesis of araC protein itself.
| 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 |
| Frato, Katherine E; Schleif, Robert F (2009) A DNA-assisted binding assay for weak protein-protein interactions. J Mol Biol 394:805-14 |
| 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 |
| 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 |
