This project seeks to elucidate how an extracellular signal changes specific gene transcription. The Escherichia coli uhp locus encodes an active transport system, UhpT, for uptake of sugar phosphates and related substrates. Transcription of the uhpT gene is induced by extracellular glucose-6-phosphatte through the action of an unusual two-component regulatory system encoded by the uhpABC genes. Our overall goal is to understand the mechanisms of transmembrane signaling by the UhpBC genes. Complex, the regulation of phosphate transfer to the transcription activator UhpA, the effect of phosphorylation of UhpA on its function, the mechanism of transcription activation by UhpA, and the basis for the co-dependent activation of transcription by UhpA and the global regulatory protein CAP. 1. The segments of UhpB that are involved in recognition of UhpA, and phosphate transfer to and from it, will be probed by genetic and biochemical tests. The role of sequestration of UhpA on the proper control of gene expression will be analyzed. The basis for the inactivity of the isolated kinase segment as auto-kinase will be explored as a function of the exposure of the site of phosphorylation, and the effect of mutations and the presence of UhpC on conformation of the phosphorylation domain. 2. The interactions of the membrane-bound UhpB and UhpC proteins in response to the presence of inducer will be examined by chemical crosslinking studies. The reconstitution of purified full-length UhpB into proteoliposomes, with and without UhpC, will allow biochemical examination of the inducer- specific control of phosphate transfer reactions by UhpB. The role of the hydrophobic N-terminal half of UhpB in membrane anchorage, binding of UhpC, and signal transduction process, by deletion or replacement of individual transmembrane segments. 3. The UhpC protein will be purified and reconstituted into proteoliposomes for assay of inducer binding and transport. Genetic screens will analyze the role of segments of UhpC in inducer binding and specificity. Chimeric proteins containing portions of UhpC and of the transporter UhpT will be made to identify the protein segments that differentiate between the signaling activity of UhpC and the transport activity of UhpT. These studies should provide new information on transmembrane signal transduction coupled between two membrane proteins.
