The Escherichia coli RhaS Transcriptional Activator: Transcriptional Activation by the DNA-Binding Domain, The Interdomain Effector Response, and Negative Autoregulation

Abstract

The chapters herein are the accepted manuscript versions of articles that were published independently in scholarly research journals. They have been combined and submitted in fulfillment of the thesis requirement for a Master of Arts degree in Microbiology from the University of Kansas Department of Molecular Biosciences. In addition to the work presented here, my graduate work included two additional projects: a high-throughput screen to identify inhibitors of the Escherichia coli RhaS protein, and a site-directed mutagenesis screen to better understand the molecular mechanisms of the L-rhamnose response in RhaS. The high-throughput screen identified a compound that inhibits DNA binding by RhaS, the related E. coli RhaR protein and the virulence activators Rns from enterotoxigenic E. coli and VirF from Shigella flexneri, but by neither E. coli LacI nor CRP. It appears that this compound may have broad, specific inhibitory activity against AraC-family proteins, making it a candidate for development into an antimicrobial drug that functions by blocking the expression of certain bacterial virulence factors that require an AraC-family activator for expression. The compound likely binds in a pocket between the two helix-turn- helix motifs of the conserved AraC-family DNA-binding domain, thereby sterically prohibiting the protein from binding DNA. In order to better understand the molecular mechanism by which the L-rhamnose signal is transmitted through RhaS from the N-terminal effector-binding domain to the C-terminal DNA-binding domain to regulate DNA binding in response to effector, I constructed a library of several dozen site-directed RhaS mutants. The goal of this work was to identify amino acids key to interdomain signaling by identifying point mutants with phenotypes consistent with defects in signaling. I focused my mutagenesis on regions of the protein predicted to be important in signaling, based on molecular modeling and similarities with related proteins. I isolated mutants in the DNA-binding domain with nearly wild-type activity (-)L-rhamnose and reduced activity (+)L-rhamnose, consistent with a decreased ability to stimulate activity (+)L-rhamnose, at positions Asn174 and Leu175. We conclude that these two residues are likely important in the signal transduction pathway; future work will identify the region of the N-terminal domain involved in this interaction.