GENETIC STUDIES OF GLUTAMINE SYNTHETASE IN BACTERIA

该项目有三个目标。 第一是详细阐述 细菌增强子结合蛋白NTRC激活的机制 由RNA的选择性全酶形式引发的转录 聚合酶，sigma 54-全酶。 二是研究感知 控制NTRC功能的电路，以响应 结合氮素，第三是阐明生理作用 在氮代谢的两个中心中间体谷氨酸和 谷氨酰胺和它们的前体2-酮戊二酸。 拟议的研究将 有助于理解转录增强子， 增强子结合蛋白一般，这些是关键的正常 代谢和人类和其他真核生物的发展。他们进一步 将有助于理解氮调节，氮调节， 以及整合这两种主要的代谢调节机制的机制 电路. NTRC通过DNA从其远端增强子位点接触σ 54全酶 环的形成，必须水解ATP，使聚合酶， 在促进剂下从封闭的络合物异构化为开放的络合物。 具体目标 关于第一个目标的是：a）开发用于 NTRC和聚合酶之间的蛋白质-蛋白质接触介导开放 复合物的形成并定义这些接触; B）确定是否 ATP水解需要热力学和动力学-- 也就是说，平衡的位置是否有利于封闭复合体; c） 为了确定能量耦合的机制是否取决于 NTRC构象之间的转变或瞬时掺入 磷酸盐转化为聚合酶，如通过监测构型所评估的 d）确定在ATP的γ磷周围的步骤; 通过ATP水解控制全部异构化为开放复合物， 特别是，它是否是初始封闭的异构化 复合物到“第二或中间”封闭复合物。 具体目标 关于第二个目标是：a）开发一种可靠的方法， 测量2-酮戊二酸盐池，如我们测量 B）确定glnA的体内速率是否 转录受谷氨酰胺/2-酮戊二酸的比例控制， 如果是，在整个转录范围内，这个比率有多大变化 rates. 关于第三个目标的具体目标是： 假设肠道细菌认为外部氮限制 谷氨酰胺的内部库大小的限制; B）确定谷氨酰胺的 谷氨酸池在谷氨酸调节中的作用。 低谷氨酸的肌肉拉伤 池在中等和高外部渗透压下缓慢生长。 是 在这种情况下它们体内的pH值异常偏碱性是他们 K+池低？他们的海藻糖池是否升高？能否 渗透保护剂脯氨酸和甜菜碱替代（K+） 谷氨酸盐？

This project has three goals. The first is to elucidate in detail the mechanism by which the bacterial enhancer-binding protein NTRC activates transcription initiation by the alternative holoenzyme form of RNA polymerase, sigma54-holoenzyme. The second is to study the sensing circuit that controls NTRC function in response to availability of combined nitrogen, and the third is to elucidate the physiological roles of the two central intermediates of nitrogen metabolism, glutamate and glutamine, and their precursor 2-oxoglutarate. 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. NTRC contacts sigma54-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. Specific aims with respect to the first goal are: a) to develop assays for the protein-protein contacts between NTRC and polymerase that mediate open complex formation and to define these contacts; b) to determine whether the requirement for ATP hydrolysis is thermodynamic as well as kinetic -- that is, whether the position of equilibrium favors closed complexes; c) to determine whether the mechanism for energy coupling depends on a transition between conformations of NTRC or on transient incorporation of phosphate into polymerase, as assessed by monitoring the configuration around the gamma phosphorus of ATP; d) to determine what step in the overall isomerization to open complexes is controlled by ATP hydrolysis, in particular, whether it is the isomerization of initial closed complexes to "second or intermediate" closed complexes. Specific aims with respect to the second goal are: a) to develop a reliable method for measuring 2-oxoglutarate pools, as we have done for measuring pools of glutamate and glutamine; b) to determine whether the in vivo rate of glnA transcription is controlled by a ratio of glutamine/2-oxoglutarate and, if so, how much this ratio varies under the full range of transcription rates. Specific aims with respect to the third goal are: a) to test the hypothesis that enteric bacteria perceive external nitrogen limitation as a limitation in the internal pool size of glutamine; b) to determine the role of the glutamate pool in osmoregulation. Srains with low glutamate pools grow slowly at intermediate and high external osmolarities. Is their internal pH abnormally alkaline under these conditions? Are their K+ pools low? Are their pools of trehalose elevated? Can the osmoprotectants proline and glycine-betaine substitute for (K+) glutamate?