This research is aimed at elucidation of the means by which the five operons which comprise the sn glycerol 3-phosphate (glp) regulon of Escherichia coli are negatively controlled by the proteins encoded by the glpEGR operon. The glpR gene encodes a repressor protein which interacts specifically with operator sites on DNA in each of the operons of the regulon. The glpE- and glpG- encoded proteins are needed together with the repressor to achieve efficient repression. This type of regulatory system containing three components has not been described previously in prokaryotes. Results of the proposed research will provide molecular details regarding the interaction of repressor with operator DNA, and will reveal the roles of the GlpE and G1pG proteins in repression. In addition, the mechanism of action of the repression system in each of the operons of the regulon will be defined in greater detail. Specifically, the role of repressor-mediated DNA looping and of integration host factor (IHF) will be determined. The objectives will be achieved by using genetic and biochemical approaches. The structure and function of the DNA-binding domain of the repressor will be studied through characterization of altered forms of the repressor generated by site-directed mutagenesis. The roles of G1pE in repression will be determined by isolation and characterization of mutants altered in G1pE and/or G1pG action, and by isolation and characterization of the G1pE and G1pG proteins. The role of IHF-binding sites and of additional potential operators in repression will be defined by determining repression in mutants deficient in IHF, and by identification of these sites on the DNA. All of the results will provide molecular details regarding the mechanism of action of this "three-component" regulatory system. This knowledge will shed light on how the cell uses specific DNA- protein interactions to respond to changes in the supply of nutrients.
