The overall long-term goals of this project are to understand the functions of the bacterial enhancer-binding protein NtrC (nitrogen- regulatory protein C) in activating transcription by the sigma 54- holoenzyme from its distant enhancer sites by DNA loop formation and must hydrolyze ATP to allow the polymerase to isomerize from closed to open complexes at a promoter. The requirement for ATP hydrolysis is thermodynamic as well as kinetic and therefore there must be a mechanism to couple the energy available to a change in conformation of polymerse-promoter complexes. To hydrolyze ATP, NtrC must be phosphorylated on an aspartic acid residue in its amino-terminal domain. This domain acts positively on the central, catalytic domain of the protein and can be thought of as the 'switch' that controls the 'motor'.
The specific aims with respect to NtrC function are: a) to study the effect of phosphorylation on communication between the N- terminal and central domains of NtrC; b) to establish an assay(s) for cyclic conformational changes in NtrC upon ATP hydrolysis; c) to establish an assay(s) for contact between NtrC and sigma 54 holoenzyme; c) to expand our collection of NtrCrepressor proteins, those that fail uniquely in positive control but not phosphorylation or DNA-binding, and test all such proteins for subfunctions of the central domain; 5) to continue structural studies of NtrC.
Specific aims with respect to metabolism are: a) to determine whether a ratio of glutamine/2-oxoglutarate controls the rate of glnA transcription in vivo; b) to determine whether the glutamine pool is depleted in nitrogen- limited B. Subtilis as it is in enteric bacteria, 3) to further characteriz the consequences of K= glutamate depletion in glnE mutant strains to determine whether there are compensatory increases in polyamines and whether turgor is grossly aberrant; 4) to determine whether protein- DNA interactions are altered in mutant strains with low K= glutamate, particularly whether protein-DNA affinity is usually high. The proposed studies will contribute to an understanding of transcriptional enhancers and enhancer-binding proteins generally, these being critical to the normal metabolism and development of humans and other eukaryotes. Further, they will contribute to understanding of nitrogen regulation, osmoregulation, and mechanisms for integrating these two major metabolic regulatory circuits.
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