Structure-Function Relationships in E. coli Response Regulators NarL and NarP

本项目的目标是利用控制细菌硝酸盐呼吸的NarQ/NarX/NarL/ NarP系统，了解基因表达双组分调控系统的分子机制。在这个系统中，NarQ和NarX蛋白感知周围环境中硝酸盐或亚硝酸盐的存在，并通过磷酸化特定的天冬氨酸来激活反应调节分子NarL或NarP。激活的反应调节因子随后与DNA上的特定位点结合，适当地开启和关闭基因。该项目已开始与一个成功的晶体结构分析的NarL。下一步计划的项目有：(1)在不同的晶体环境中完成NarL结构，研究晶体环境效应；(2)相似但独立作用的NarP的x射线结构分析；(3) NarL的DNA结合c端结构域及其DNA识别序列的结构分析；(4)抗去磷酸化和与DNA强结合的NarL和NarP组成突变体的检测；(5)全NarL/DNA和NarP/DNA复合物的x射线分析。***本研究的意义在于，它将首次在分子水平上对双组分传感器/响应系统进行系统研究，其结果适用于细菌双组分控制系统的广泛进化范围。它还将阐明高等生物中的多组分遗传控制系统，这些系统也通过磷酸化关键效应蛋白起作用。总的来说，NarQ/NarX/NarL/NarP家族包括一个复杂但灵活的控制系统，可以根据硝酸盐和亚硝酸盐的相对浓度调节几个基因的相对表达。这个分子反应网络的四组分例子背后的完整逻辑尚不清楚。增加对它如何工作以及为什么它必须如此复杂的理解是这个项目的主要目标。% % %

9630809 Dickerson The goal of this project is to understand the molecular mechanism of two-component regulatory systems of genetic expression, using the NarQ/NarX/NarL/ NarP system that controls nitrate respiration in bacteria. In this system, NarQ and NarX proteins sense the presence of nitrate or nitrite in the surrounding environment, and activate response regulator molecules NarL or NarP by phosphorylating a particular aspartic acid. The activated response regulators then bind to specific sites on DNA, turning genes on and off as appropriate. The project has begun with a successful crystal structure analysis of NarL. Further projects planned are: (1) Completion of the NarL structure in a different crystal environment, to examine crystal environment effect; (2) X-ray structure analysis of the similar but independently-acting NarP; (3) Structure analysis of the DNA-binding C-terminal domain of NarL in a complex with its recognition sequence of DNA; (4) Examination of constitutive mutants of NarL and NarP that resist dephosphorylation and bind strongly to DNA; and (5) X-ray analysis of full NarL/DNA and NarP/DNA complexes. *** The significance of this study is that it will be the first systematic investigation at the molecular level of a two component sensor/response system, with results applicable to the broad evolutionary range of bacterial two-component control systems. It will also shed light on multi- component genetic control systems in higher organisms, that also act by phosphorylating key effector proteins. Taken together the NarQ/NarX/NarL/NarP family comprises a complex but flexible control system that can adjust the relative expression of several genes in response to the relative concentrations of nitrate and nitrite. The full logic behind this four-component example of a molecular response network is not yet understood. An increased understanding of how it works and why it must be so complex is the major goal of this project. %%%