GENETIC STUDIES OF GLUTAMINE SYNTHETASE IN BACTERIA

这个项目有三个目标。首先是详细阐明 细菌增强子结合蛋白NTRC的激活机制 RNA的另一种全酶形式启动转录 聚合酶，Sigma54-全酶。二是对传感技术的研究 响应于可用性而控制NTRC功能的电路 结合氮素，三是阐明其生理作用 在氮代谢的两个中心中间体中，谷氨酸和 谷氨酰胺及其前体2-氧戊二酸。拟议的研究将 有助于理解转录增强子和 增强子结合蛋白一般，这些对正常的 人类和其他真核生物的代谢和发育。此外，他们还 将有助于理解氮的调节，渗透调节， 以及整合这两个主要代谢调节的机制 电路。 NTRC通过DNA从其远端的增强子位点接触sigma54-全酶 形成环路，必须水解三磷酸腺苷才能使聚合酶 在启动子上从封闭的复合体异构化为开放的复合体。具体目标 关于第一个目标是：a)开发用于 NTRC和聚合酶之间介导开放的蛋白质-蛋白质接触 复杂的形成和定义这些接触；b)确定是否 ATP水解的要求是热力学的，也是动力学的-- 也就是说，均衡的位置是否有利于闭合复合体；c) 要确定能量耦合的机制是否取决于 NTRC构象之间的转换或在瞬时并入时 磷酸盐转化为聚合酶，通过监测构型进行评估 围绕着ATP的伽马磷；d)确定在 开放络合物的总异构化受ATP水解的控制， 特别是，无论是异构化还是初始封闭 从复合体到“第二或中间”的封闭复合体。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-羟基戊二酸盐池，就像我们测量 谷氨酸和谷氨酰胺；b)测定GlnA在体内的百分率 转录受谷氨酰胺/2-羟基戊二酸的比例控制， 如果是这样的话，在整个转录范围内，这一比例变化有多大？ 费率。关于第三个目标的具体目标是：a)测试 肠道细菌将外部氮素限制感知为 谷氨酰胺内部池大小的限制；b)确定 谷氨酸池在渗透调节中的作用。低谷氨酸的燕麦 在中等和较高的外部渗透压下，水池增长缓慢。是 它们体内的pH值在这些条件下异常碱性吗？是他们的 K+池低？它们的海藻糖池是不是升高了？能不能 渗透保护剂脯氨酸和甘氨酸甜菜碱替代(K+) 谷氨酸？

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?