9727745 Nixon The Rhizobium melitoti dicarboxylate transport (dct) system and the Salmonella typhimurium nitrogen regulation (ntr) system are partly understood signal transduction systems. Each one utilizes a two-component transduction mechanism to regulate the active state of a sigma54 dependent transcriptional activator (DctD and NtrC, respectively). Despite some similarities, it is clear that two diverse physical strategies are used to integrate two-component signal transduction with sigma54 dependent transcription activation. DctD is controlled negatively, with its ATPase domain being actively repressed by the two-component receiver domain until the latter is phosphorylated; in contrast, NtrC is controlled positively, with the phosphorylated form of its two-component receiver domain being needed for the ATPase domain to display its activity. The long-term objective is to understand the biochemical mechanisms of two-component signal transduction through the DctD and NtrC proteins to the RNA polymerase-sigma54 dependent promoter complexes with which they interact. The immediate goal is to begin determining how regulated self-assembly controls the active state of these sigma54 dependent transcriptional activators. Specific aims include: 1. quantifying the oligomerization properties of native DctD, constitutively active DctD variants, fragments 1-127, 1-143, and 1-384 of these proteins, NtrC, and NtrC-P; 2. determining if and to what extent the oligomerization of DctD is affected by the molecules with which it is known to interact: ATP, specific DNA binding sites, sigma54, and the (-subunit of core RNA polymerase; 3. determining if there is a correlation between cooperativity and oligomerization properties for DctD, DctD-P, and constitutively active variants of DctD, using DNAse I footprinting to measure free energies of intrinsic DNA binding and cooperativity, and comparing these results with those of objectives 1 and 2; and 4. identifyi ng mutants of dctD that encode DctD substitutions affecting cooperative but not intrinsic DNA binding, both for the transcriptionally inactive and active states of the protein, and assessing whether or not those residues are also important for oligomerization. In order to respond to changes in their environment, organisms use a "two-component" signal transduction mechanism, which results in differences in gene expression. This kind of response is ubiquitous in eubacteria, and has also been characterized in eukaryotes and archaebacteria. The purpose of this project is to investigate transcription factor proteins that cause changes in gene expression when they are phosphorylated in response to environmental changes. Following phosphorylation, they may bind cooperatively with each other and with DNA, to bring about changes in gene expression. The two systems under study are the dicarboxylic acid transporter from Rhizobium meliloti, which is required for nitrogen fixation, and the nitrogen regulation system from Salmonella typhimurium. This project will study the detailed interaction of these transcription factors with each other and with DNA in response to environmental changes.
