Structure/function analysis of a bacterial protein kinase/ phosphatase

细菌中氮对基因表达的调控是通过转录因子氮调节因子I （NRA）的可逆磷酸化来完成的，NRA与特定的DNA序列结合，当被磷酸化时，激活转录。NRA的磷酸化和去磷酸化是由双功能蛋白激酶/磷酸盐氮调节剂II （NRII）催化的。本项目概述了旨在阐明NRII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有关，统称为双组分系统，它们共享一个涉及蛋白质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有几个被充分证明的活性：蛋白质与ATP结合并催化磷酸化基团，然后在NRA催化的反应中转移到NRA。最后，在另一种被称为PII的信号转导蛋白的存在下，NRII催化磷酸化形式的NRA的去磷酸化。Ninfa博士计划通过经典诱变或寡核苷酸定向位点特异性诱变来识别导致体内氮调节基因调节改变的NRII的改变。然后对改变后的NRII蛋白进行纯化，并确定这些改变对NRII已知活性的影响。通过这种方式，预计可以阐明每项活动及其控制所需的NRII的结构特征。此外，NRII的激酶和磷酸盐活性在控制氮调控基因中的作用可以进一步描述。另一个将采取的方法是更完整地描述野生型NRII的结构和活动。例如，将对磷酸酶活性的测定进行潜在有用的改进，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，不同的基因在特定的时间以一种适合有机体有序发育或对压力情况作出有效反应的方式表达。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很感兴趣，并且正在研究一种从细菌开始的开关模型。细菌对氮饥饿的反应是开启某些基因的表达。该过程所需的开关组件已经确定。最重要的开关成分是一种叫做氮调节器II的蛋白质。这种蛋白质已经被纯化，尼法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（另一种称为氮调节剂I的蛋白质）和PII（另一种蛋白质）相互作用。为了获得这样的信息，将在已知的地点进行微妙的扰动，并对属性的变化进行监测。最具信息量的是那些只影响NRII属性的扰动，而不影响其他属性。在这个项目中，这种细微的扰动对NRII的影响将被非常详细地研究，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. ***