Regulation of Microbial Nitrogen Metabolism

微生物氮代谢的调节

• 批准号: 6681026
• 负责人: ROBERT A BENDER
• 金额: $ 34.43万
• 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 2007-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6681026
• 关键词: bacterial genetics; biological signal transduction; electron microscopy; gel electrophoresis; gel mobility shift assay; genetic promoter element; genetic regulatory element; genetic transcription; glutamate dehydrogenase; microorganism metabolism; mutant; nitrogen metabolism; operon; polymerase chain reaction; protein structure function

DESCRIPTION (provided by applicant): The Nitrogen Assimilation Control Protein, NAC, is a transcriptional regulator that activates dozens of operons in K. pneumoniae, one of which (hut) is used to degrade histidine as a nitrogen source. NAC also represses several genes, including gdhA (encoding glutamate dehydrogenase) and its own gene, nac. Previous work from Dr. Bender's lab shows that NAC uses several different mechanisms to regulate transcription, some using dimers and some tetramers of NAC. Indeed, NAC is one of the most versatile transcriptional regulators known. Dr. Bender and his colleagues propose a detailed genetic analysis of these modes of regulation. Their overall hypothesis is that the DNA sequence of the NAC-binding site determines the conformation of NAC, at hut tetramer formation is inhibited, at gdhA it is permitted, and at nac it is greatly stimulated. The genetic analysis of activation at hut will include "positive control" mutants (that can repress but not activate), mutants frozen in activating conformation whatever the DNA sequence, mutants that fail to interact with RNAP, and mutants that fail to dimerize. Repression at gdhA will include a further analysis of "negative control" mutants (that can activate but not repress), as well as a test of the DNA looping model to explain the requirement for two well-separated sites for repression. Other models will also be considered. The role of the DNA sequence of the NAC site at the nac gene will be investigated to establish its role in tetramer formation and to contrast it to the site at gdhA. Although NAC-mediated repression of gdhA is the key focus of this proposal, Dr. Bender will also explore the positive effectors that are required for gdhA expression and their interaction with NAC. In particular, a lysine-sensitive positive effector of gdhA expression will be characterized and used as a model for the characterization of other positive effectors of gdhA.

描述(申请人提供)：氮同化控制蛋白，NAC，是一种转录调节因子，能激活肺炎克雷伯菌中的数十个操纵子，其中一个(HUT)被用来作为氮源降解组氨酸。NAC还抑制几个基因，包括gdhA(编码谷氨酸脱氢酶)和它自己的基因nac。本德尔博士的实验室之前的工作表明，NAC使用几种不同的机制来调节转录，其中一些使用了NAC的二聚体和一些四聚体。事实上，NAC是已知的最多功能的转录调控因子之一。本德尔博士和他的同事们提议对这些调控模式进行详细的基因分析。他们的总体假设是，NAC结合位点的DNA序列决定了NAC的构象，但在四聚体形成被抑制，在gdhA被允许，在NAC被极大地刺激。HUT对激活的遗传分析将包括“阳性对照”突变体(可以抑制但不能激活)，冻结在激活构象中的突变体，无论DNA序列如何，不能与RNAP相互作用的突变体，以及不能二聚体的突变体。在gdhA的抑制将包括对“阴性对照”突变体(可以激活但不能抑制)的进一步分析，以及对DNA循环模型的测试，以解释对两个分离良好的抑制位点的要求。其他型号也将被考虑。将研究Nac基因上Nac位点的DNA序列的作用，以确定其在四聚体形成中的作用，并将其与gdhA处的位点进行比较。虽然NAC介导的抑制GdhA是这项提议的主要焦点，但Bender博士也将探索gdhA表达所需的积极效应及其与NAC的相互作用。特别是，赖氨酸敏感的gdhA表达的正效应将被表征，并被用作表征gdhA的其他正效应的模型。