Signal Transduction by Histidine Kinases and their Response Regulators

描述（由申请人提供）：组氨酸激酶是真核丝氨酸/苏氨酸和酪氨酸蛋白激酶的细菌对应物，它们都是细胞应答和适应外部信号（应激）所需的信号转导。典型的细菌组氨酸激酶（不包括参与趋化的组氨酸激酶）是跨膜信号转导，调节其同源反应调节因子的磷酸化状态，其主要功能是转录因子。这反过来调节（激活或抑制）适应压力所需的特定基因。与丝氨酸/苏氨酸和酪氨酸蛋白激酶不同，在信号转导过程中，一个高能磷酸化基团通过组氨酸激酶从ATP传递到反应调节因子。因此，组氨酸激酶反应调节系统被称为“双组分His-Asp磷接力信号转导系统”。其中，来自大肠杆菌的EnvZ-OmpR系统是研究最广泛的系统之一。EnvZ是一种渗透敏感的组氨酸激酶，其胞质激酶结构域的核磁共振三维结构已经被确定。在Aim 1中，我们将继续对细胞膜周围受体结构域和细胞质连接体结构域进行结构研究，以了解膜中EnvZ二聚体的整体三维结构。我们还将在EnvZ和Tar（一种趋化性Asp传感器）之间构建混合信号传感器，以获得通过信号输入调节组氨酸激酶活性的精确分子机制。在Aim 2中，我们将破译磷酸化的OmpR （OmpR- p）与OmpR启动子中六个OmpR- p结合位点的精确结合模式，以证明从初步结果中预测的一个称为“飞驰模型”的模型。我们还将通过RT-PCR分析OmpR-P转录水平和micF和micC两种反义rna翻译水平对ompF和ompC外膜孔蛋白基因的相互调控机制。

DESCRIPTION (provided by applicant): Histidine kinases are the bacterial counterpart to eukaryotic Ser/Thr and Tyr protein kinases, all of which function as signal transducers required for cellular response and adaptation to external signals (stresses). Typically bacterial histidine kinases (excluding a histidine kinase involved in chemotaxis) are transmembrane signal transducers that regulate the phosphorylation state of their own cognate response regulators, which function mostly as transcription factors. This in turn regulates (activates or represses) a specific gene required for adaptation to the stresses. In contrast to Ser/Thr and Tyr protein kinases, a high-energy phosphoryl group is relayed from ATP through a histidine kinase to a response regulator during signal transduction. Therefore, the histidine kinase-response regulator systems are referred as "the two-component His-Asp phosphorelay signal transduction system". Of these, the EnvZ-OmpR system from Escherichia coli is one of the most extensively investigated systems. EnvZ is an osmo-sensing histidine kinase and the NMR three-dimensional structures of its cytoplasmic kinase domains have been determined. In Aim 1, we will continue the structural studies on the periplasmic receptor domain and the cytoplasmic linker domain to comprehend the global three-dimensional structure of the EnvZ dimer in the membrane. We will also construct hybrid signal transducers between EnvZ and Tar, a chemotaxis Asp sensor to obtain mechanistic insights into the precise molecular mechanism of regulation of the histidine kinase activity by a signal input. In Aim 2, we will decipher the precise mode of binding of phosphorylated OmpR (OmpR-P) to six OmpR-P binding sites in the ompF promoter to prove a model called "galloping model" predicted from the preliminary results. We will also analyze the reciprocal regulatory mechanism for the ompF and ompC outer membrane porin genes at the level of transcription by OmpR-P and at the level of translation by two antisense RNAs, micF and micC RNAs by RT-PCR.