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

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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. %%%