The AbrB protein of Bacillus subtilis regulates a number of genes, including some essential for the sporulation process, that are expressed during the transition state between balanced exponential growth and stationary phase. It can act as either a positive or negative regulator of transcription, depending upon the particular target gene. It is a DNA-binding protein with specificity toward the promoters it controls. The determinants of this specificity are largely unknown but appear to be related to some type of three-dimensional DNA structure that can be assumed by a variety of different base sequences. We propose to examine the AbrB-DNA interaction in greater detail with the goal to understand more fully how AbrB discriminates between the promoters it controls and those it does not. These studies will include obtaining detailed In vitro footprints of AbrB binding to DNA and correlating this information to the intracellular situation by the use of in vivo footprinting techniques where possible. We will also isolate mutant binding sites and construct in vitro deletion and insertion variants to further delimit elements necessary for recognition. Second site mutants affecting AbrB activity will be searched for in order to more fully understand any other factors that impinge upon AbrB-mediated regulation. Where possible, we will assay the effects that other regulatory proteins may have upon AbrB activity. As one possible mechanism for AbrB-mediated regulation is through its effect upon the localized structure of the promoter, we will examine the effects of AbrB binding upon changes in DNA bending. We will also initiate a crystallographic analysis of the AbrB protein with the ultimate goal of relating structure to function.
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