This project seeks to elucidate the mechanisms by which a signal outside the cell leads directly to changes in transcription activity at a gene. The Escherichia coli uhp locus encodes an active transport system UhpT for the uptake of a variety of sugar phosphates and related organophosphates. Transcription of the uhpT gene is induced by the presence of extracellular glucose 6-phosphate (Glu6P), but not by internally generated Glu6P, through the action of three regulatory proteins encoded by the upstream uhpABC locus. Our goal is to understand in molecular detail the mechanism of transmembrane regulation of uhpT transcription by a phospho-relay cascade. The UhpA and UhpB proteins are members of an underexplored subfamily of the widespread group of bacterial two-component regulatory proteins, and have several unusual features. The UhpC protein is involved in transmembrane signaling but is related in structure and sequence to the UhpT family of transport proteins.
The specific aims for the requested period of support fall into four areas. 1. The DNA sites for binding of UhpA and the global regulator CAP will be defined, in terms of their sequence determinants, stoichiometry and cooperative of protein binding, and interaction with RNA polymerase. 2. The portions of the UhpA protein that participate in determining DNA binding and transcription activation will be determined by genetic techniques, and the effect of the phosphorylation of UhpA on its oligomeric state, DNA binding affinity, and effect on transcription will be studied through biochemical approaches. 3. The portions of UhpB that are involved in auto-phosphorylation, transfer of phosphate to UhpA, and stimulation of phosphate release from UhpA will be probed by directed mutagenesis. 4. The interactions of the membrane-embedded UhpB and UhpC proteins that comprise the transmembrane signaling complex will be determined by isolation of allele-specific suppressors of mutants with altered regulation, by affinity chromatography, and by reconstitution of the regulatory system in membrane vesicles. Chimeric proteins combining portions of UhpC and the UhpT transport protein will attempt to identify portions of these homologous proteins that contribute to transport and signaling functions, and to substrate specificity. It is anticipated that completion of the above aims will provide deeper insight into the action of an unusual regulatory system, but one which operates in essence in the control of many bacterial virulence factors.
