GENETIC AND BIOCHEMICAL STUDIES OF PHOB ACTIVATION

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DESCRIPTION: The long term objective of the proposed research is to understand how phosphorylation activates response regulator proteins. These proteins control a wide range of adaptive responses in both prokaryotes and eukaryotes. This proposal focuses on the activation of PhoB, a response regulator from E. coli that controls gene expression in response to external phosphate levels. PhoB is made up of two domains: an N-terminal regulatory domain that contains the site of phosphorylation, and a C-terminal transcriptional activator domain that binds DNA and interacts with RNA polymerase. PhoB is a molecular switch that is turned on by auto-phosphorylation and off by dephosphorylation. The regulatory domain of PhoB is composed of two functional modules: a switch and a relay. The switch catalyzes phosphorylation and dephosphorylation whereas the relay transmits the phosphorylation status of the switch to the rest of the protein. Control of the phosphate response occurs by regulation of PhoB phosphorylation and dephosphorylation. To understand the activation of PhoB, this research will study PhoB autophosphorylation and dephosphorylation, will characterize the activated state of PhoB and will test competing hypotheses regarding the mechanism by which phosphorylation of the regulatory domain increases the activity of the C-terminal output domain. The enzymatic mechanisms of PhoB will be investigated through the analysis of mutant proteins. Site directed mutations will be created in PhoB to probe the roles of specific residues. In addition, a genetic screen for randomly generated mutations will be used to identify residues that are involved in the activation of PhoB. Fluorescence spectrophotometric and 32P-based assays will be used to follow PhoB phosphorylation and dephosphorylation. The activated state of PhoB will be characterized in the absence of auxiliary proteins by using a low molecular weight phospho-donor, acetyl phosphate. DNA binding, DNA bending, and PhoB:PhoB interactions will be studied using multiple gel retardation and footprinting assays. Experiments to test how phosphorylation of the regulatory domain of PhoB activates the effector domain involve the generation and analysis of several chimeric CheY/PhoB constructs. We will also employ gel-filtration chromatography and fluorescence spectroscopy to follow potential oligomerization and/or conformational changes within PhoB that occur upon phosphorylation. The proposed work is important for understanding the molecular mechanisms of signal transduction. In particular, it will help elucidate general principals regarding the activation of proteins by phosphorylation. Phosphorylation-activated transcription factors, similar to PhoB, are widespread throughout nature and control responses that regulate the cell cycle, differentiation, development, virulence, metabolite utilization, and transport. Knowledge gained about the mechanisms of PhoB activation should be readily applicable to other systems.