Structure/function analysis of a bacterial protein kinase/ phosphatase

细菌蛋白激酶/磷酸酶的结构/功能分析

• 批准号: 9318792
• 负责人: Alexander Ninfa
• 金额: $ 28.66万
• 依托单位: Regents of the University of Michigan - Ann Arbor
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 1994
• 资助国家: 美国
• 起止时间: 1994-04-01 至 1998-03-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=9318792&HistoricalAwards=false
• 关键词: Structure; function; analysis; bacterial; protein

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. ***

NINFFA 9318792对细菌基因表达的氮调节是通过转录因子氮调节因子I(Nra)的可逆磷酸化完成的，该转录因子氮调节因子与特定的dna序列结合，当磷酸化时，激活转录。NRA的磷酸化和去磷酸化是由双功能蛋白激酶/磷酸盐氮调节因子II(NRII)催化的。本项目概述了旨在阐明NRII的激酶和磷酸盐活性及其控制的机制的实验。NRA和NRII与大量的细菌调节蛋白有关，统称为二组分系统，它们共享一个保守的信号转导机制，涉及蛋白质磷酸化和去磷酸化。从NRII的研究中获得的信息将增加我们对相关的激酶/磷酸盐蛋白的了解。NRII有几个已知的活性：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，在另一种称为PII的信号转导蛋白存在的情况下，NRII催化磷酸化形式的NRA的去磷酸化。宁法博士计划通过经典的突变或寡核苷酸指导的定点突变来识别NRII的变化，这些变化导致体内氮调控基因的调节发生变化。然后将对改变的NRII蛋白进行纯化，并将确定这些改变对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氮调控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全面地描述野生型NRII的结构和活性。例如，将采用潜在的USEF ul改进磷酸酶活性的测定，并计划NRII的结晶和结构分析。活着的生物体并不总是表达其DNA中存在的所有基因；相反，各种基因在特定的时间以适合有机体有序发育或有效应对应激情况的方式表达。基因的开启和关闭是由分子开关完成的。PI对这种分子开关如何发挥作用感兴趣，并正在研究一种来自细菌的模型开关。细菌对氮饥饿的反应是通过启动某些基因的表达。已确定此过程所需的交换机组件。最重要的开关成分是一种名为氮调节因子II的蛋白质。这种蛋白质已经被提纯，宁法博士正在研究它的性质。特别是，他想知道NRII蛋白的哪些部分负责与另一种被称为氮调节蛋白I的NRA和另一种蛋白质PII相互作用。为了获得这样的信息，将在已知地点进行微妙的干扰，并将监测物业的变化。最能提供信息的是那些只影响NRII属性的扰动，而不影响其他属性。在这个项目中，我们将非常详细地研究这种微扰对NRII的影响，以增加我们对这种分子开关如何工作的理解。***