The long term goal of this project is to achieve a better understanding of how external signals are communicated to the machinery regulating gene expression in the cell. Our focus, with this goal in mind, has been a study of the bgl operon in E.coli, which is regulated by a two-component system consisting of a sensory protein, present in the cytoplasmic membrane and a regulatory protein, present in the cytoplasm. The sensor, termed BglF, is a bifunctional protein which transports substrate and which also acts as a protein kinase and phosphatase, regulating the activity of the regulator, BglG. In the absence of substrate, BglF phosphorylates BglG thus inactivating it, while in the presence of substrate, BglF dephosphorylates BglG, resulting in its activation. The active, non-phosphorylated form of BglG, is a dimer which binds to nascent RNA transcripts acting to prevent termination of transcription. During the next grant period we will define regions of contact between BglF and BglG that are essential for phosphorylation. This will be done by isolating and characterizing BglF mutants that fail to recognize and thus phosphorylate BglG. Such mutants will in turn be used to isolate BglG suppressors which will define the regions of contact between the two proteins. As part of this effort, we will identify the specific histidine that is phosphorylated in BglG. A major part of our effort will be a structure-function analysis of BglG. Residues crucial to RNA binding will be defined by site specific mutagenesis and also by selection of novel RNA sequences, produced by randomization of chosen segments of the RNA target. We will attempt to determine the crystal structure of the RNA binding domain bound to its target RNA. We will locate and characterize the sequences essential for BglG dimerization with the aim of understanding how phosphorylation blocks dimer formation. We will use in vitro approaches to reproduce the antitermination reaction in vitro. We wish to know whether BglG is sufficient for antitermination, if transcriptional pausing is essential for its action and precisely when the RNA target becomes accessible to the protein.
