RUI:Transcriptional Activation by the Bacillus Subtilis ComK Protein

Williams College, Williamstown MA

Investigators

Abstract

The objective of this research is to elucidate the mechanism by which the Bacillus subtilis competence transcription factor, ComK, activates transcription. Competence in B. subtilis is characterized by the cell's ability to bind and take up exogenous DNA. In addition to controlling its own expression, ComK is essential for the expression of the proteins that comprise the DNA binding and uptake system and for the competence-related expression of the recombination genes recA and addAB. ComK binds in the minor groove to sequences upstream of the -35 region and causes bending of the DNA. Three classes of ComK-regulated promoters are distinguished based on the spacing between ComK dimer binding sites: In the first class (recA, addAB, dinA, and nucA) the interval between dimer binding sites is 2 helical turns; the second class, comprising the late competence genes (comC, -G, -E, and -F), contain binding sites separated by 3 helical turns; and the third class contains a single representative, the comK promoter, in which the binding sites are separated by 4 helical turns. The downstream binding sites for all ComK-dependent promoters are located in regions proposed for the binding of the alpha subunits of E. coli RNA polymerase, which also binds in the minor groove. The PI will characterize the DNA binding domain of ComK and test the hypothesis that ComK affects the binding of the a subunits of RNA polymerase. The ComK DNA binding domain will be characterized using a combination of mutational analysis, NMR structural determination, and molecular modeling. The PI will determine whether or not ComK interacts with RNA polymerase alpha when it binds to representatives of the three classes of ComK-dependent promoters, recA, comG, and comK, by a combination of hydroxyl radical footprinting, targeted footprinting, protein-protein crosslinking, and mutational analyses. DNA binding and transcriptional activation will be assessed for wild-type and mutant ComK proteins using mobility shift assays, in vitro transcription assays, and analyses of transcriptional activation of recA-lacZ, comG-lacZ, and comK-lacZ fusions. This research involves investigating the way in which the ComK protein turns on the expression of an assortment of genes that enable the bacterium Bacillus subtilis to take up external DNA and incorporate this DNA into its chromosome. The results should elucidate how ComK controls the expression of dozens of genes that are required when Bacillus subtilis cells enter a specialized state for genetic exchange. Since the action of ComK likely involves interactions with RNA polymerase, the enzyme that governs gene expression in all bacteria, this research should further the understanding of bacterial gene regulation in general.