Genome analysis suggests that two-component phosphotransfer is the predominant mechanism used by prokaryotes to couple changes in the environment to changes in gene expression or protein function. In E. coli, the EnvZ-OmpR two-component system regulates transcription of the outer membrane porin genes ompF and ompC, and the csg locus (required for biofilm formation), in response to changes in medium osmolarity. EnvZ is an inner-membrane histidine kinase that phosphorylates the response regulator OmpR. The promoter architectures of the ompF, ompC and csg loci differ, and it is likely that OmpR-DNA and OmpR-RNA polymerase interactions are distinct at each promoter. This proposal uses biochemical approaches to study post-phosphorylation events in the response regulator OmpR. Aim 1 seeks to identify how phosphorylation of the N-terminus of OmpR results in a conformational change in and subsequent stimulation of DNA binding by its C-terminus. Aim 2 probes the OmpR-DNA interactions at the ompF, ompC and csg promoters to identify the OmpR structural elements and DNA bases required for each interaction. The last aim examines the interaction of the alpha subunit of RNA polymerase with both OmpR and OmpR-specific promoter DNA to determine the role of alpha at OmpR-dependent promoters.