The overall objective of this work has been to understand how the enteric bacteria, especially Klebsiella aerogenes, respond to the quality and quantity of their nitrogen source. The research proposed here is tightly focused on the Nitrogen Assimilation Control Protein (NAC), a LysR-type regulator that activates transcription of ammonia-generating operons like hutUH (histidine utilization) and represses transcription of ammonia-using operons like gdhA (glutamate dehydrogenase). There are 3 specific aims of this proposal. First, a genetic analysis of positive-control (PC) and negative-control (NC) mutants will be carried out. PC mutants will be selected here as those that bind DNA (at the nac promoter) normally but fail to activate hutUp and NC are those that bind but fail to repress at gdhAp. Such mutations will include mutant sites that cannot deform NAC appropriately, mutant NAC that has altered site recognition, and mutant NAC that has altered surfaces required for protein-protein contact. Second, the effect of NAC on the binding and activity of RNAPolymerase will be determined using NAC, PC and NC mutants of NAC, and small (active) fragments of NAC. These will be tested using gel-shift assays (for binding) and run- off transcription assays with normal RNAP and a mutant form with a deletion of the a-CTD. finally the structure of NAC will be probed using chimeras formed between NAC and other LysR proteins (OxyR and CysB). The turnover rate of NAC will be measured and tested to see if it is regulated. And an attempt will be made to confirm whether the N- and C-termini of NAC are in close proximity. If time allows, a system to study oriented heterodimers will also be developed. An attempt to grow crystals of NAC or to purify large quantities of NAC fragments for nmr solution will continue throughout the proposal period. By the end of these 5 years, the data gathered should give hints on 1. how NAC activates transcription and the role of the DNA in causing this activation, 2. how NAC represses transcription (whether there is a NAC-NAC interaction involved and whether the DNA site plays a role in it), and 3. what parts of the NAC polypeptide are involved in communicating among the various interaction sites.
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