9513275 Kenney All organisms must communicate effectively with their environment and adapt in order to survive. In both prokaryotes and eukaryotes, a paradigm for adaptive responses is the two-component regulatory systems. A two-component system contains a sensor (which is often a membrane protein which functions in trans-membrane signaling) and a response regulator (often a DNA binding protein which regulates transcription). Changes in the environment result in the phosphorylation of the sensor from intracellular ATP. The sensor then transfers its phosphoryl group to the regulator which alters its output. The sensor (or sometimes another distinct protein) stimulates the dephosphorylation of the regulator to re-set the system. The two-component system in E. coli regulates the porin genes by responding to changes in osmolarity of the growth medium. At low osmolarity, ompF is preferentially expressed and at high osmolarity, it is repressed and ompC expression is switched on. EnvZ, the sensor, transfers its phosphoryl group onto OmpR, a DNA binding protein that regulates transcription of the porin genes. Little is known about how the OmpR-like proteins interact with DNA; they do not contain sequences similar to those of DNA binding domains found in other regulators. A major goal of this work is to precisely determine the region of OmpR which contacts DNA and identify the amino acids which make direct contacts. Studies will also address the dual role of OmpR as an activator (of both ompF and ompC) and also as a repressor (ompF only). A model arising from genetic studies predicts that phospho-OmpR (OmpR-P) binds with high affinity to activate ompF and at low affinity to repress ompF and activate ompC. It also predicts that unphosphorylated OmpR plays no role in transcriptional regulation. These studies will provide the first direct biochemical evaluation of this hypothesis. Measurements of DNA binding using fluorescence anisotropy will examine the effect of phospho-OmpR on binding to flu oresceinated promoter fragments. In addition, biochemical approaches will be employed to investigate the potential regulatory involvement of OmpR-P-induced oligomerization, determine the region of OmpR which interacts with the promoters as well as RNA polymerase. ***
