Structure/function analysis of a bacterial protein kinase/ phosphatase

Ninfa 9318792细菌中基因表达的氮调节是通过转录因子氮调节因子I（NRA）的可逆磷酸化来实现的，所述转录因子氮调节因子I（NRA）结合特定的DNA序列，并且当磷酸化时激活转录。 NRA的磷酸化和去磷酸化由双功能蛋白激酶/磷酸盐氮调节剂II（NRII）催化。 该项目概述了旨在阐明NRII的激酶和磷酸盐活性及其控制机制的实验。 NRA和NRII与大量细菌调节蛋白相关，统称为双组分系统，其共享涉及蛋白磷酸化和去磷酸化的保守信号转导机制。 从NRII的研究中获得的信息将增加我们对相关激酶/磷酸盐蛋白的理解。 NRII具有几种有据可查的活性：该蛋白结合ATP并催化磷酸化基团，然后在NRA催化的反应中转移到NRA。 最后，在另一种称为PII的信号转导蛋白的存在下，NRII催化NRA的磷酸化形式的去磷酸化。 Ninfa博士计划通过经典诱变或阿托伐他汀定点诱变来鉴定导致体内氮调节基因调节改变的NRII改变。 然后将纯化改变的NRII蛋白，并确定改变对NRII已知活性的影响。 通过这种方式，预计可以阐明每种活动所需的NRII结构特征及其控制。 此外，NRII的激酶和磷酸酶活性在氮调节基因调控中的作用也可以进一步阐明。 将采取的另一种方法是更完整地表征野生型NRII的结构和活性。 例如，将采用磷酸酶活性测定的潜在有用改进，并计划对NRII进行结晶和结构分析。 生物体并不总是表达其DNA中存在的所有基因;相反，各种基因在某些时候以适合生物体有序发育或有效应对压力的方式表达。 基因的开启和关闭是由分子开关完成的。 PI对这种分子开关的功能感兴趣，并正在研究细菌的模型开关。 细菌对氮饥饿的反应是开启某些基因的表达。 已确定此过程所需的开关组件。 最重要的开关成分是一种叫做氮调节剂II的蛋白质。 这种蛋白质已经被纯化，Ninfa博士正在研究它的特性。 特别是，他想知道NRII蛋白的哪些部分负责与NRA（另一种称为氮调节因子I的蛋白质）和PII（另一种蛋白质）相互作用。 为了获得这些信息，将在已知的地点进行微妙的扰动，并对属性的更改进行监测。 信息量最大的是那些只影响NRII性质而不影响其他性质的扰动。 在这个项目中，将非常详细地研究这种微妙的扰动对NRII的影响，以增加我们对这种分子开关如何工作的理解。 ***

Ninfa 9318792 Nitrogen regulation of gene expression in bacteria is accomplished by the reversible phosphorylation of the transcription factor Nitrogen regulator I (NRA) which binds to specific DNA sequences and, when phosphorylated, activates transcription. The phosphorylation and dephosphorylation of NRA are catalyzed by the bifunctional protein kinase/phosphates Nitrogen regulator II (NRII). This project outlines experiments designed to elucidate the mechanisms of the kinase and phosphates activities of NRII and their control. NRA and NRII are related to a large number of bacterial regulatory proteins, known collectively as the two- component systems, that share a conserved mechanism of signal transduction involving protein phosphorylation and dephosphorylation. The information gained from the studies of NRII will increase our understanding of the related kinase/phosphates proteins. NRII has several well documented activities: The protein binds to ATP and catalyzes the phosphorylation group is then transferred to NRA in a reaction catalyzed by NRA. Finally, in the presence of another signal transduction protein known as PII, NRII catalyzes the dephosphorylation of the phosphorylated form of NRA. Dr. Ninfa plans to identify, by classical mutagenesis or oligonucleotide-directed site-specific mutagenesis, alternations of NRII that result in altered regulation of nitrogen-regulated genes in vivo. The altered NRII proteins will then be purified and the effect of the alterations on the known activities of NRII will be ascertained. In this way it is anticipated that the structural features of NRII required for each of the activities and their control can be elucidated. In addition, the roles of the kinase and phosphates activities of NRII in the control of nitrogen- regulated genes can be further delineated. Another approach that will be taken is to more completely characterize the structure and activities of wild-type NRII. For example, a potentially usef ul improvement in the assay for the phosphatase activity will be employed and the crystallization and structural analysis of NRII are planned. %%% Living organisms do not express all of the genes present in their DNA at all times; rather, various genes are expressed at certain times in a manner that is appropriate for the orderly development of the organism or for an effective response to a stressful situation. The turning on and off of genes is accomplished by molecular switches. The PI interested in how such molecular switches function, and is studying a model switch from bacteria. Bacteria respond to nitrogen starvation by turning on the expression of certain genes. The components of the switch required for this process have been identified. The most important switch component is a protein called Nitrogen Regulator II. This protein has been purified, and Dr. Ninfa is investigating its properties. In particular, he would like to know which parts of the NRII protein are responsible for interacting with NRA, another protein known as Nitrogen Regulator I, with PII, another protein. In order to obtain such information, will be subtly perturbed at known sites, and alterations in properties will be monitored. Most informative are those perturbations that affect only property of NRII, leaving the other properties intact. In this project, the effects of such subtle perturbations to NRII will be studied in great detail in order to increase our understanding of how this molecular switch works. ***