Regulatory Mechanisms of the E. coli Arabinose Operon

描述(申请人提供)：本项目的长期目标是尽可能全面和深入地了解大肠杆菌中L-阿拉伯糖操纵子的调控。短期内，以下概述的问题将得到解决。从长远来看，就像在所有研究中一样，目标是了解重要的监管现象，以便我们可以将这些知识用于健康或医疗问题，并使用相同的原则设计和建立其他系统。我将重点放在阿拉伯糖操纵子及其调控蛋白AraC上，因为它是少数几个密集研究的模型系统之一。它显示了各种转录调控现象，同时也足够简单，可以采用遗传、生理、体外、生化和生物物理的方法。 实验将在两个一般领域进行。主要工作将是用核磁共振确定AraC和相关蛋白质或蛋白质复合体的DNA结合域的结构。这些包括：与或不与调控的N-末端臂结合的DNA结合域，当与紧密结合的DNA位点和弱结合位点结合时的结构，以及在臂和DNA结合域中加强或减弱臂结合的一些突变体的结构。我们还将调查一些机械问题。这些将集中在调控臂和AraC DNA结合域之间的相互作用。对于野生型调控臂和结构域以及一些突变体，将测量手臂对结构域的亲和力。我们还将寻找仅在抑制状态下起作用的二聚化结构域之间的额外相互作用。我们将确定Trpr和TetR通过调节小分子配体来调节其DNA结合亲和力的机制。调节臂与其他蛋白质结合。当手臂不与其他蛋白质或结构域结合时会发生什么？它会排斥其他蛋白质吗？我们将研究这一问题，以及在此基础上建立监管机制的可能性。

DESCRIPTION (provided by applicant): The long-term objective of this project is to understand as fully and as deeply as possible regulation of the L-arabinose operon in the bacterium Escherichia coli. In the short term, questions as outlined below will be addressed. In the longer term, as in all research, the objective is to understand important regulatory phenomena so that we may use this knowledge in health or medical issues and also design and build other systems using the same principles. I am focusing on the arabinose operon and its regulatory protein AraC because it is one of a small number of intensively studied model systems. It displays a variety of transcriptional regulatory phenomena while at the same time is sufficiently simple as to allow genetic, physiological, in vitro, biochemical, and biophysical approaches. Experiments will be done in two general areas. The main body of work will be the determination by NMR of the structure of the DNA binding domain of AraC and related proteins or protein complexes. These include: the DNA binding domain with and without the regulatory N-terminal arm bound to it, the structures when bound to a tight-binding DNA site and a weak-binding site, and the structures of a number of mutants in the arm and in the DNA binding domain that strengthen or weaken binding of the arm. We will also investigate a number of mechanistic questions. These will be centered around the interaction between the regulatory arm and the AraC DNA binding domain. The affinity of the arm for the domain will be measured for wild type regulatory arm and domain and for a number of mutants. We will also look for an additional interaction between the dimerization domain that functions only in the repression state. We will determine the mechanisms by which TrpR and TetR regulate their DNA binding affinity in response to regulating small molecule ligands. Regulatory arms bind to other proteins. What happens when an arm does not bind other proteins or domains? Does it repel other proteins? We will examine this question and the possibility of developing a regulatory mechanism based upon it.