REGULATION OF MICROBIAL NITROGEN METABOLISM

这项工作的总体目标是了解肠道细菌，特别是产气克雷伯氏菌，如何对其氮源的质量和数量做出反应。这里提出的研究紧紧围绕着氮同化控制蛋白(NAC)，这是一种LysR类型的调节蛋白，它激活组氨酸利用等氨产生操纵子的转录，并抑制GdhA(谷氨酸脱氢酶)等氨产生操纵子的转录。这项提议有三个具体目标。首先，对阳性对照(PC)和阴性对照(NC)突变体进行遗传分析。PC突变体将被选为那些正常结合DNA(在nac启动子上)但不能激活hutUp的突变体，NC是那些在gdhAp结合但未能抑制的突变体。这些突变将包括不能适当变形NAC的突变位点，改变位点识别的突变NAC，以及改变蛋白质-蛋白质接触所需表面的突变NAC。其次，将利用NAC的NAC、PC和NC突变体以及NAC的小(活性)片段来确定NAC对RNA聚合酶结合和活性的影响。这些将使用凝胶移位分析(结合)和与正常RNAP和a-CTD缺失的突变形式的径流转录分析进行测试。最后，将利用NAC和其他LysR蛋白(OxyR和CysB)之间形成的嵌合体来探索NAC的结构。将对NAC的周转率进行测量和测试，以确定是否受到监管。并将尝试确认NAC的N-末端和C-末端是否非常接近。如果时间允许，还将开发一个研究定向异二聚体的系统。在整个建议期内，将继续尝试生长NAC晶体或提纯大量NAC碎片用于核磁共振溶液。到这5年结束时，收集到的数据应该会为以下问题提供线索：1.NAC如何激活转录以及DNA在导致这种激活中的作用；2.NAC如何抑制转录(是否存在NAC-NAC相互作用以及DNA位点是否在其中发挥作用)，以及3.NAC多肽的哪些部分参与了不同相互作用位点之间的通信。

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.