Gene Control in Infection and Lysogeny by Phage Lambda

描述（由申请人提供）：这个项目的目的是了解RNA聚合酶对RNA合成延伸期的控制，这是转录调控的一个关键方面。我们将利用一种特异性的抗终结者蛋白，即噬菌体λ的Q蛋白，它在基因组特异性位点成为RNA聚合酶的亚基，从而通过防止转录终止来允许下游基因的表达。这种蛋白质提供了一个高度明确和可访问的系统，以获得控制转录伸长的普遍酶促过程的机制见解；例如，HIV病毒的TAT蛋白是转录延伸的调节剂，在许多方面与lambda Q蛋白和其他细菌抗终止剂一样起作用。对这些机制的详细了解将使治疗方法依赖于特定分子制剂的设计。我们将学习一种结构修饰的本质，它被命名为一种屏障，是lambda Q蛋白在RNA聚合酶中诱导的，目的是使其对终止子不敏感。我们将通过生化和遗传分析来研究转录延伸因子NusA在构建屏障中的作用。我们将使用突变分析来发现lambda Q蛋白修饰RNA聚合酶的位点和途径，特别是了解Q如何调节转录暂停这一RNA聚合酶的普遍功能行为。这项工作将补充我们继续努力了解终止机制本身。我们还将研究蛋白Mfd的作用机制，该蛋白介导转录偶联DNA修复过程，并在招募DNA修复蛋白时解离停滞的延伸复合物。了解ATP的能量是如何被Mfd利用的，将有助于阐明终止的机制和终止所涉及的能垒。利用细菌和噬菌体模型系统，该项目有助于了解遗传调控的基本机制，这是发现疾病分子基础的重要任务。它还直接研究了与HIV发病机制和有毒细菌感染相关的重要调控途径。

DESCRIPTION (provided by applicant): The objective of this project is to understand control of the elongation phase of RNA synthesis by RNA polymerase, a critical facet of transcription regulation. We will utilize a specific antiterminator protein, the Q protein of bacteriophage lambda, which becomes a subunit of RNA polymerase at a genome-specific site and thereby allows expression of downstream genes by preventing transcription termination. This protein provides a highly defined and accessible system with which to obtain mechanistic insights into universal enzymatic processes that control transcription elongation; for example, the TAT protein of the HIV virus is a regulator of transcription elongation that acts in many ways like lambda Q protein and other bacterial antiterminators. A detailed understanding of these mechanisms will allow approaches to therapies that depend upon the design of specific molecular agents. We will learn the nature of a structural modification, named a barrier that lambda Q protein induces in RNA polymerase in order to make it insensitive to terminators. We will study the role of the transcription elongation factor NusA in constructing the barrier, through biochemical and genetic analysis. We will use mutational analysis to discover the sites and pathways of modification of RNA polymerase by lambda Q protein, in particular to understand how Q regulates transcription pausing, a universal functional behavior of RNA polymerase. This work will complement our continuing efforts to understand the mechanism of termination itself. We also will study the mechanism of action of the protein Mfd, which mediates the process of transcription-coupled DNA repair, and acts to dissociate stalled elongation complexes as it recruits DNA repair proteins. Understanding how the energy of ATP is used by Mfd will illuminate the mechanism of termination and the energetic barriers involved in termination. PUBLIC HEALTH RELEVANCE Using bacterial and bacteriophage model systems, this project contributes to understanding basic mechanisms of genetic regulation, an undertaking essential to discovering the molecular basis of disease. It also directly investigates regulatory pathways related to those essential to pathogenesis by HIV and infection by toxic bacteria.