GENETIC STUDIES OF GLUTAMINE SYNTHETASE IN BACTERIA

本项目的总体长期目标是了解 细菌增强子结合蛋白NtrC（氮- 调节蛋白C）通过σ 54- 全酶通过DNA环形成从其远端增强子位点分离， 必须水解ATP，使聚合酶从封闭的 在启动子处打开复合物。 ATP水解的要求 是热力学的也是动力学的，因此 将可用能量与构象变化相结合的机制 聚合促进剂复合物。 为了水解ATP，NtrC必须 氨基末端天冬氨酸残基磷酸化 域 该结构域对中心催化结构域起积极作用 可以被认为是控制蛋白质的“开关”， “发动机”。 关于NtrC职能的具体目标是： 研究磷酸化对N- B）建立测定NtrC末端和中心结构域的方法， ATP水解时NtrC的环状构象变化; c） 建立NtrC和sigma 54之间接触测定 全酶; c）扩大我们收集的NtrC阻遏蛋白， 在阳性对照中唯一失败但不磷酸化的那些， DNA结合，并测试所有这些蛋白质的中枢神经系统的子功能。 5）继续进行NtrC的结构研究。 具体目标， 关于代谢的是：a）确定是否有 谷氨酰胺/2-酮戊二酸控制glnA在体内的转录速率; B）确定谷氨酰胺库是否耗尽氮- 有限B。尽管它在肠道细菌中存在，3）为了进一步表征 在glnE突变株中K=谷氨酸耗竭的后果， 确定多胺是否有代偿性增加， 是否肿胀严重异常; 4）确定蛋白质- 在具有低K=谷氨酸的突变株中，DNA相互作用发生改变， 特别是蛋白质-DNA亲和力是否通常很高。 的 建议的研究将有助于理解转录 增强子和增强子结合蛋白，这些是关键的 对人类和其他人的正常新陈代谢和发育的影响 真核生物 此外，它们将有助于了解氮 监管，可持续监管，以及整合这两者的机制 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.