Gene Control in Infection and Lysogeny by Phage Lambda

描述(申请人提供)：这个项目的目标是了解RNA聚合酶对RNA合成的延伸阶段的控制，RNA聚合酶是转录调控的一个关键方面。我们将利用一种特定的抗终止子蛋白，即噬菌体lambda的Q蛋白，它成为基因组特定位置的RNA聚合酶的一个亚单位，从而通过阻止转录终止来允许下游基因的表达。该蛋白提供了一个高度明确和可访问的系统，用于从机制上深入了解控制转录延长的通用酶过程；例如，HIV病毒的Tat蛋白是转录延长的调节因子，其作用方式与lambda Q蛋白和其他细菌抗终止物有许多相似之处。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，称为lambda Q蛋白在RNA聚合酶中诱导的屏障，以使其对终止子不敏感。我们将通过生化和遗传分析，研究转录延伸因子NusA在构建屏障中的作用。我们将利用突变分析来发现lambda Q蛋白修饰RNA聚合酶的位置和途径，特别是了解Q是如何调节转录暂停的，这是RNA聚合酶的一种普遍功能行为。这项工作将补充我们理解终止机制本身的持续努力。我们还将研究MFD蛋白的作用机制，它介导转录偶联DNA修复过程，并在招募DNA修复蛋白时解离停滞的伸长复合体。了解MFD如何利用ATP的能量将有助于阐明MFD的终止机制以及终止过程中涉及的能量势垒。利用细菌和噬菌体模型系统，该项目有助于理解基因调控的基本机制，这项工作对于发现疾病的分子基础至关重要。它还直接调查与艾滋病毒致病和有毒细菌感染相关的调控途径。

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.