NARL IN NITRATE DEPENDENT GENE EXPRESSION

反应调节蛋白NarL是双组分信号转导系统调节回路的一员，介导硝酸盐对大肠杆菌厌氧呼吸基因表达的调节。杆菌 本研究计划分析NarL反应调节蛋白的N-末端和C-末端结构域的调节特征，以确定它们在DNA结合特异性、亲和力和转录激活/抑制中的作用。 所要解决的问题包括：NarL与DNA的结合是否是协同的; NarL的DNA结合特异性是否存在于蛋白质的C端结构域，如果存在，NarL的转录调节特性是否也位于C端; N端结构域是否在NarL的DNA结合亲和力中起作用;最后，对于各种NarL调节的靶序列，NarLWT、NarLWT-磷酸盐和NarL的C-末端结构域的Kon和Koff是什么，以及调节序列的突变分析是否改变速率常数。使用这两种体外方法（DNA酶I足迹分析、滤膜结合测定、凝胶位移分析、定点诱变等）并将采用体内表达研究来研究这些问题。 从拟议的研究中获得的信息不仅将扩大我们对NarXQLP硝酸盐调控系统的了解，而且还将增强我们对涉及细菌生长和存活的许多方面的全球双组分调控系统家族的了解。

The response regulator protein, NarL, is a member of a regulatory circuit involving dual two-component signal transduction systems that mediate nitrate regulation of anaerobic respiratory gene expression in E. coli. This research proposal analyzes the regulatory characteristics of the N-terminal and C-terminal domains of the NarL response regulatory protein to determine their roles in DNA binding specificity, affinity and transcriptional activation/repression. The issues that will be addressed include whether DNA binding by NarL is cooperative; whether the DNA-binding specificity of NarL resides in the C- terminal domain of the protein, and if so, are the transcriptional regulatory properties of NarL also located in the C-terminus; whether the N-terminal domain plays a role in the DNA binding affinity of NarL; and finally, what is the Kon and Koff of NarLWT, NarLWT-Phosphate and the C-terminal domain of NarL for various NarL-regulated target sequences and does mutational analyses of the regulatory sequences alter the rate constants. The use of both in vitro (DNase I footprint analysis, filter binding assays, gel shift analysis, site-directed mutagenesis, etc.) and in vivo expression studies will be employed to investigate these questions. The information gained from the proposed research will not only broaden our knowledge of the NarXQLP nitrate-regulatory systems, but will also enhance our knowledge of the global family of two component regulatory systems involved in many aspects of bacterial growth and survival.