Regulatory Mechanisms of the E. coli Arabinose Operon

描述（由申请人提供）：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。在短期内，将处理下列问题。从长远来看，与所有研究一样，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，并使用相同的原理设计和构建其他系统。我专注于阿拉伯糖操纵子及其调节蛋白AraC，因为它是少数深入研究的模型系统之一。它显示了多种转录调控现象，同时足够简单，以允许遗传，生理，体外，生物化学和生物物理方法。 实验将在两个方面进行。工作的主体将是通过NMR确定AraC和相关蛋白质或蛋白质复合物的DNA结合结构域的结构。其中包括：有和没有调节的N-末端臂结合到它的DNA结合域，当结合到紧密结合的DNA位点和弱结合位点时的结构，以及臂和DNA结合域中的一些突变体的结构，这些突变体加强或削弱臂的结合。这些将集中在调节臂和AraC DNA结合结构域之间的相互作用。将测量野生型调节臂和结构域以及许多突变体的臂对结构域的亲和力。我们还将寻找仅在阻遏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调节其DNA结合亲和力以响应调节小分子配体的机制。调节臂与其他蛋白质结合。当一个臂不结合其他蛋白质或结构域时会发生什么？它排斥其他蛋白质吗？我们将研究这一问题，并研究在此基础上建立一个监管机制的可能性。

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.