Mechanism of transcript elongation control by RfaH

描述(申请人提供)：延伸是转录周期中最长的部分，在此期间，RNA聚合酶在模板上的移动受到许多障碍DNA结合蛋白、DNA损伤、终止信号等的阻碍。细菌蛋白RfaH通过增加RNA聚合酶的处理能力来调节细胞壁和被膜成分、抗生素和毒力因子的表达。RfaH的作用依赖于一种名为Ops的DNA序列，它在伸长过程中介导RfaH向RNA聚合酶的募集。在第一个授予期间，我们获得了RfaH的X射线结构，确定了它在转录复合体上的结合位置，表征了RfaH在不同调节位置和延伸特性改变的酶上的作用，并表明RfaH通过防止停顿而不是通过增加核苷酸添加速度来发挥作用。这一机制很可能在其他抗终止剂中基本保守。在这项提案中，我们将使用生化、遗传和结构方法的组合来解决RfaH作用的几个方面。本项目的第一个目标是研究RfaH的作用机制。我们将结合遗传、生化和结构分析来剖析N末端结构域(足以进行RfaH抗暂停活性)与转录延伸复合体的相互作用，并阐明由这些相互作用引发的构象变化。该项目的第二个目标是阐明业务处人员在招聘RfaH方面的作用。我们认为OPs不仅与RfaH建立碱基特异的联系，而且还诱导RfaH结合所需的特殊的皱缩DNA构象。该项目的第三个目标是测试“调制”的C-末端结构域在RfaH招募后是否改变了它的结构，并参与了与翻译装置的串扰。该项目的第四个目标是通过体内交联法和染色质免疫沉淀法分别鉴定RfaH相关蛋白和基因来表征RfaH调节子。我们还将通过定量RT-PCR分析选定的RfaH操纵子。公共卫生相关性：该项目旨在阐明转录因子RfaH调节基因表达的机制。RfaH基因存在于人类、昆虫和植物病原体中；此外，rfaH对于动物模型的毒力是必不可少的。这些研究将揭示RfaH的作用机制，阐明其DNA靶点在转录调控中的独特作用，并识别可能是未知毒力因子的细胞RfaH靶点。

DESCRIPTION (provided by applicant): Elongation is the longest part of transcription cycle during which RNA polymerase movement along the template is hindered by many roadblocks DNA-bound proteins, DNA lesions, termination signals, etc. Factors that allow RNA polymerase to bypass these barriers are required for efficient synthesis of long RNAs in all domains of life. Bacterial protein RfaH regulates expression of the cell wall and capsule components, antibiotics, and virulence factors by increasing the RNA polymerase processivity. RfaH action depends on a DNA sequence called ops that mediates RfaH recruitment to RNA polymerase during elongation. In the first granting period, we obtained the X-ray structure of RfaH, identified its binding site on transcription complex, characterized RfaH effects at different regulatory sites and on enzymes with altered elongation properties, and showed that RfaH acts by preventing pausing rather than by increasing the rate of nucleotide addition. This mechanism is likely fundamentally conserved in other antiterminators. In this proposal, we will use a combination of biochemical, genetic, and structural approaches to address several aspects of RfaH action. The first goal of this project is to study the mechanism of RfaH action. We will use a combination of genetic, biochemical, and structural analyses to dissect interactions of the N-terminal domain (which is sufficient for RfaH anti-pausing activity) with the transcription elongation complex and to elucidate the confomational changes triggered by these interactions. The second goal of this project is to elucidate the role of the ops element in recruitment of RfaH. We propose that ops not only establishes base-specific contacts with RfaH but also induces a specialized scrunched DNA conformation that is required for RfaH binding. The third goal of this project is to test if the "modulatory" C-terminal domain changes its structure after RfaH recruitment and is involved in cross-talk with the translation apparatus. The fourth goal of this project is to characterize the RfaH regulon by identifying the RfaH-associated proteins and genes by in vivo crosslinking and chromatin immuno-precipitation, respectively. We will also analyze selected RfaH operons by quantitative RT PCR. PUBLIC HEALTH RELEVANCE: This project aims to elucidate the mechanism by which transcription factor RfaH regulates gene expression. The rfaH genes are present in human, insect, and plant pathogens; moreover, RfaH is essential for virulence in animal models. These studies will reveal the mechanism of RfaH action, elucidate the unique role of its DNA target site in transcriptional control, and identify cellular RfaH targets which may be uncharacterized virulence factors.