REGULATION OF MICROBIAL NITROGEN METABOLISM

这项研究的总体目标是了解基因表达是如何 在全球调节信号如氮的作用下被激活 饥饿 特别是，这项研究侧重于如何不同的 阳性激活剂NAC和CAP-cAMP（对氮和碳反应 饥饿分别）可以各自激活来自相同的 启动子，组氨酸利用操纵子的hutU启动子。 NAC对动力学参数（速率常数和产率常数）的影响 用于RNA聚合酶的转录起始。 这些影响将是 与NAC激活的嵌合hut-lac启动子相比， 从lacZ启动子转录。 同样，CAP-cAMP的作用 将确定对hutU启动子的作用，以与NAC进行比较， 解释hutU中两个不同CAP结合位点的作用 启动子 一组平行的实验将检验 NAC和RNA聚合酶之间，特别是集中在C- a亚基的末端结构域。 此外，NAC抑制某些基因转录的能力 (e.g. ghdA基因）将被检查，以解释为什么抑制NAC 似乎比激活更复杂 NAC的结构 蛋白质本身将通过物理方法（例如， 结晶学和NAC的最小活性结构域的鉴定） 和遗传学方法（以确定与DNA，RNA相互作用的结构域） 聚合酶等）。 最后，该项目将导致持续改善 用于产气克雷伯氏菌遗传分析的工具， λ噬菌体溶原性的一种新模式和一种新形式的分析 不依赖于氮调节。

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.