REGULATION OF MICROBIAL NITROGEN METABOLISM

微生物氮代谢的调节

• 批准号: 6386287
• 负责人: ROBERT A BENDER
• 金额: $ 32.09万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 1979
• 资助国家: 美国
• 起止时间: 1979-05-01 至 2003-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6386287
• 关键词: DNA directed RNA polymerase; Klebsiella; bacterial genetics; crystallization; gel electrophoresis; gel mobility shift assay; genetic promoter element; genetic regulatory element; genetic transcription; microorganism metabolism; nitrogen metabolism; nuclear magnetic resonance spectroscopy; nuclear runoff assay; operon; protein structure function

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.

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，如何响应其氮源的质量和数量。这里提出的研究主要集中在氮同化控制蛋白（NAC）上，这是一种LysR型调节剂，可以激活氨生成操纵子（如hutUH（组氨酸利用））的转录，并抑制氨使用操纵子（如gdhA（谷氨酸脱氢酶））的转录。该提案有三个具体目标。首先，将对阳性对照（PC）和阴性对照（NC）突变体进行遗传分析。PC突变体在此将被选择为正常结合DNA（在nac启动子处）但不能激活hutUp的那些，NC是结合但不能抑制gdhA β的那些。这样的突变将包括不能使NAC适当变形的突变位点、具有改变的位点识别的突变NAC和具有改变的蛋白质-蛋白质接触所需的表面的突变NAC。其次，NAC对RNA聚合酶的结合和活性的影响将使用NAC的NAC、PC和NC突变体以及NAC的小（活性）片段来确定。这些将使用凝胶位移测定（用于结合）和用正常RNAP和具有α-CTD缺失的突变形式的径流转录测定来测试。最后，将使用NAC和其它LysR蛋白（OxyR和CysB）之间形成的嵌合体探测NAC的结构。新来港定居人士的流动率将被衡量和测试，以确定是否受到监管。并尝试确认NAC的N-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。在整个提案期间，将继续尝试生长NAC晶体或纯化大量NAC片段用于核磁共振溶液。到这5年结束时，收集到的数据应能提示1. NAC如何激活转录以及DNA在引起这种激活中的作用，2. NAC如何抑制转录（是否涉及NAC-NAC相互作用以及DNA位点是否在其中起作用），以及3. NAC多肽的哪些部分参与各种相互作用位点之间的通信。