My lab focusses on understanding molecular mechanisms of sensory transduction to the transcriptional apparatus in prokaryotes. We examine the genetics and biochemistry of C4-dicarboxylate transport in Rhizobium meliloti and Rhizobium leguminosarum that fix nitrogen in a symbiotic relationship with the agriculturally important legumes alfalfa and peas. Because dicarboxylic acids are used as energy sources to fuel such nitrogen fixation, the research may lead to improved strains of rhizobium.
Our working hypothesis is that two membrane proteins cooperate to detect external ligand, causing one to phosphorylate a cytoplasmic transcriptional regulator. The activated regulator binds upstream of the promoter it regulates, making contact with RNA polymerase via a DNA looping mechanism that sometimes involves an additional DNA scaffolding protein. The activator, which binds and hydrolyses ATP, then stimulates a conversion of the polymerase/promoter complex from a closed form in which DNA is double stranded to an open one containing the single stranded bubble needed for transcription to initiate.
Previous work has focussed on establishing these qualitative features of the signal transduction pathway. Current efforts are aimed at quantitative characterization of DNA binding by the activator protein and accessory scaffolding protein. Binding by the activator has been found to include a cooperativity component, which increases upon activation. We have also isolated point mutations that de-regulate the transcriptional activator, causing it to activate transcription in the absence of signal transduction. These mutations map onto a surface of the N-terminal domain of the activator protein, which we can model using NMR and crystal structure data for homologous proteins.
Future experiments are intended to understand the biochemical basis for the basal and increased cooperativity, to clarify the precise role of ATP hydrolysis in the activation process, and to characterize the physical basis for signal transduction from the N-terminal domain to the rest of the activator protein. We also collaborate with Dr. Tim Hoover (Microbiology, University of Georgia) who is examining the physical basis for interaction between the activator and the RNA polymerase.
We also collaborate with Dr. Dale Kaiser of the Department of Microbiology at Stanford to examine the role of similar activator proteins in controlling the starvation response of Myxococcus xanthus, and with Dr. Karen Miller of the Department of Food Science at Penn State to investigate the role of cyclic-a-glucans in the nodulation process that leads to effective symbiosis between rhizobium species and legumes.
54-dependent transcriptional activator,
may be negatively controlled by a subdomain in the C-terminal end of its
two-component receiver module. Mol. Microbiol. 13:51-61.
54-dependent transcriptional activator.
J. Biol. Chem. 269:20401-20409.
