Research: Molecular mechanisms of bacterial transcription activation; AraC family proteins.
My primary research interest is understanding the regulation of gene expression (especially positive regulation) at a molecular level. In our lab we examine the molecular biology of gene regulation in E. coli, with a focus on the L-rhamnose catabolic genes, and the fact that the L-rhamnose regulator genes are members of the AraC family of regulatory proteins. Many AraC family proteins are regulators of virulence factors in bacterial pathogens, therefore, we use the L-rhamnose regulators as models to understand the function of proteins in this family. Our ultimate goal is to identify small molecules that block the function of AraC family proteins and have potential as anti-bacterial agents.
The widespread availability of antibiotics since the 1940's has dramatically reduced the number of deaths due to bacterial infections, however we are rapidly losing our advantage over our bacterial foes as antibiotic resistance becomes increasingly prevalent. We must investigate novel targets for antibacterial agents to avoid the return of commonplace deaths due to bacterial infections. Many members of the very large AraC/XylS family of transcription activators are required for the expression of virulence factors in bacterial pathogens. In several cases it has been shown that deletion of an AraC/XylS activator drastically reduces pathogenesis, indicating that members of this family have potential as targets for antibacterial agents. Our long-term goal is to identify small molecule inhibitors of AraC/XylS family activators. Towards this goal, we are currently working on two fronts. First, we are performing a screen of a large library of small molecules for ones that specifically inhibit the activity of the AraC/XylS family protein RhaS. Once we identify compounds that are inhibitory, we will investigate their mechanism of action. Towards this end, we are also investigating the mechanisms used by the AraC/XylS family protein RhaS that underlie its ability to activate transcription of the appropriate genes and under the appropriate conditions. Once we have identified the mechanisms of a few more of RhaS's functions, we will be in an excellent position to identify the mechanisms of action of inhibitory small molecules. Ultimately, we will screen for inhibitors of AraC/XylS family activators of virulence factors in bacterial pathogens. We expect that these inhibitors have potential to be developed into novel strategies for treatment of bacterial diseases.
We have already obtained a substantial amount of information about RhaS function. For example, we have identified four amino acid residues in RhaS that make base-specific contacts with its DNA-binding site, and further, have identified the base-pairs in the DNA that each of these residues contacts. This allows us to unequivocally orient each monomer of the RhaS dimer on its DNA site. We have also identified two amino acid residues in RhaS that are directly required to contact RNA polymerase to activate transcription. In addition, we have identified the amino acid residues in the sigma70 subunit of RNA polymerase that each of these RhaS residues contacts. There are only very few transcription activators for which interactions with RNA polymerase have been defined at this level of detail. We are currently investigating the mechanisms by which RhaS: dimerizes; binds its ligand, L-rhamnose; and transmits the information that L-rhamnose is or is not available from its N-terminal ligand binding and dimerization domain to its C-terminal DNA-binding and transcription activation domain.
- Bacterial transcriptional regulation
- Antimicrobial agents