Signal Transduction by Histidine Kinases and their Response Regulators

描述（由申请人提供）：组氨酸激酶是真核生物Ser/Thr和Tyr蛋白激酶的细菌对应物，所有这些蛋白激酶都作为细胞应答和适应外部信号（应激）所需的信号转导子发挥作用。通常，细菌组氨酸激酶（不包括参与趋化性的组氨酸激酶）是调节其自身同源反应调节剂的磷酸化状态的跨膜信号转导物，其主要作为转录因子起作用。这反过来又调节（激活或抑制）适应压力所需的特定基因。与Ser/Thr和Tyr蛋白激酶相反，在信号转导过程中，高能磷酰基从ATP通过组氨酸激酶传递到反应调节剂。因此，组氨酸激酶反应调节系统被称为“双组分His-Asp磷酸化信号转导系统”。其中，来自大肠杆菌的EnvZ-OmpR系统是最广泛研究的系统之一。EnvZ是一种对组氨酸敏感的组氨酸激酶，其胞质激酶结构域的NMR三维结构已被确定。在目标1中，我们将继续对周质受体结构域和胞质接头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。我们还将构建EnvZ和Tar之间的混合信号转导器，一种趋化性Asp传感器，以获得对信号输入调节组氨酸激酶活性的精确分子机制的机械见解。目的2：我们将破译磷酸化OmpR（OmpR-P）与ompF启动子中的6个OmpR-P结合位点的精确结合模式，以证明根据初步结果预测的称为“奔马模型”的模型。我们还将通过RT-PCR分析ompF和ompC外膜孔蛋白基因在OmpR-P转录水平和两种反义RNA micF和micC RNA翻译水平上的相互调节机制。

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.