GENETIC STUDIES OF GLUTAMINE SYNTHETASE IN BACTERIA

该项目的总体长期目标是了解 细菌增强子结合蛋白NTRC的功能 调节蛋白C)通过Sigma 54激活转录- 全酶通过DNA环的形成从其远端的增强子位置和 必须水解三磷酸腺苷才能使聚合酶从封闭状态异构化 在启动子上打开复合体。三磷酸腺苷水解酶的要求 既是热力学的，也是动力学的，因此必须有一个 将可用能量与构象变化相耦合的机制 聚合-促进剂复合体。要分解ATP，NTRC必须 在其氨基末端的天冬氨酸残基上磷酸化 域。这个结构域对中心的催化结构域起正作用 并且可以被认为是控制 “马达”。与NTRC职能有关的具体目标是：a) 研究磷酸化对N-亚甲基-4-氨基丁酸之间通讯的影响 NTRC的末端和中心结构域；b)建立一种分析方法(S) ATP水解时NTRC的循环构象变化；c)至 建立NTRC与Sigma 54接触的检测方法(S) 全酶；c)扩大我们的NtrCrepressor蛋白质集合， 那些在阳性对照中唯一失败但不是磷酸化或 DNA结合，并测试所有这些蛋白质的中央亚功能 5)继续进行NTRC的结构研究。具有特定目标的 关于新陈代谢的问题是：a)确定 谷氨酰胺/2-羟基戊二酸在体内控制glnA转录的速度； B)确定谷氨酰胺池中的氮是否耗尽- 限制枯草杆菌与肠道细菌一样，3)进一步鉴定 GlnE突变株中K=谷氨酸耗竭的后果 确定多胺是否有补偿性增加和 肿胀是否严重异常；4)确定蛋白质- 在低K=谷氨酸的突变菌株中，DNA相互作用发生了变化， 尤其是蛋白质-DNA亲和力通常是否很高。这个 拟议的研究将有助于理解转录 增强子和增强子结合蛋白，这些是至关重要的 对人类和其他动物的正常新陈代谢和发育 真核生物。此外，它们将有助于理解氮 调节、渗透调节和整合这两者的机制 主要代谢调节回路。

The overall long-term goals of this project are to understand the functions of the bacterial enhancer-binding protein NtrC (nitrogen- regulatory protein C) in activating transcription by the sigma 54- holoenzyme from its distant enhancer sites by DNA loop formation and must hydrolyze ATP to allow the polymerase to isomerize from closed to open complexes at a promoter. The requirement for ATP hydrolysis is thermodynamic as well as kinetic and therefore there must be a mechanism to couple the energy available to a change in conformation of polymerse-promoter complexes. To hydrolyze ATP, NtrC must be phosphorylated on an aspartic acid residue in its amino-terminal domain. This domain acts positively on the central, catalytic domain of the protein and can be thought of as the 'switch' that controls the 'motor'. The specific aims with respect to NtrC function are: a) to study the effect of phosphorylation on communication between the N- terminal and central domains of NtrC; b) to establish an assay(s) for cyclic conformational changes in NtrC upon ATP hydrolysis; c) to establish an assay(s) for contact between NtrC and sigma 54 holoenzyme; c) to expand our collection of NtrCrepressor proteins, those that fail uniquely in positive control but not phosphorylation or DNA-binding, and test all such proteins for subfunctions of the central domain; 5) to continue structural studies of NtrC. Specific aims with respect to metabolism are: a) to determine whether a ratio of glutamine/2-oxoglutarate controls the rate of glnA transcription in vivo; b) to determine whether the glutamine pool is depleted in nitrogen- limited B. Subtilis as it is in enteric bacteria, 3) to further characteriz the consequences of K= glutamate depletion in glnE mutant strains to determine whether there are compensatory increases in polyamines and whether turgor is grossly aberrant; 4) to determine whether protein- DNA interactions are altered in mutant strains with low K= glutamate, particularly whether protein-DNA affinity is usually high. The proposed studies will contribute to an understanding of transcriptional enhancers and enhancer-binding proteins generally, these being critical to the normal metabolism and development of humans and other eukaryotes. Further, they will contribute to understanding of nitrogen regulation, osmoregulation, and mechanisms for integrating these two major metabolic regulatory circuits.