The overall goal of this research is to understand how gene expression is activated in response to global regulatory signals like nitrogen starvation. In particular, this research focuses on how to different positive activators, NAC and CAP-cAMP (responding to nitrogen and carbon starvation respectively) can each activate transcription from the same promoter, the hutU promoter of the histidine utilization operons. The effects of NAC on the kinetic parameters (rate and yield constants) for transcription initiation by RNA Polymerase. These effects will be compared with those at al chimeric hut-lac promoter where NAC activates transcription from the lacZ promoter. Similarly, the effects of CAP-cAMP on the hutU promoter will be determined both to compare with NAC and to explain the effects of two different CAP-binding sites within the hutU promoter. A parallel set of experiments will examine the interactions between NAC and RNA Polymerase, concentrating especially on the C- terminus domain of the a subunit. In addition, the ability of NAC to repress transcription of some genes (e.g. the ghdA gene) will be examined to explain why repression by NAC appears to be more complex than activation. The structure of the NAC protein itself will be probed by a combination of physical methods (e.g. crystallography and identification of the smallest active domain of NAC) and genetic approaches (to identify domains that interact with DNA, RNA Polymerase, etc). Finally this project will result in the continued improvement of the tools for genetic analysis of Klebisella aerogenes, including a study of a novel mode of lysogeny for phage lambda, and an analysis of a new form of NTR-independent nitrogen regulation.
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