Structure & Function of Prokaryotic Transcript Cleavage

结构

• 批准号: 6435140
• 负责人: SERGEI BORUKHOV
• 金额: $ 27.44万
• 依托单位: SUNY DOWNSTATE MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 1996
• 资助国家: 美国
• 起止时间: 1996-05-01 至 2005-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6435140
• 关键词: DNA directed RNA polymerase; Escherichia coli; X ray crystallography; bacterial genetics; bacterial proteins; crosslink; cysteine; gene mutation; protein protein interaction; protein structure function; transcription factor

DESCRIPTION (Provided by applicant): The prokaryotic transcript cleavage factors GreA and GreB identified in Escherichia coli are thought to have three biologically important and evolutionarily conserved functions in transcription: suppression of elongation arrest, facilitation of promoter escape, and enhancement of transcription fidelity. These functions are accomplished by the ability of Gre factors to induce cleavage of nascent RNA in the ternary transcription complex. The broad goal of this project is to understand the mechanism of action and the structure-function relationships of GreA and GreB. Four types of experiments will be carried out. #1. To identify functionally important localities of Gre factors, we will introduce by oligonucleotide directed random mutagenesis single amino acid substitution of all residues in Gre proteins except those that are involved in the intramolecular interactions. We will also introduce single and multiple amino acid substitutions in the conserved loop and in the region immediately preceding the C-terminal domain by site-directed mutagenesis. The mutants will be characterized by specific transcription assays in vivo and in vitro. #2. To elucidate interactions between Gre and other components of the TC, we will identify mutations in the beta and beta prime subunits of RNAP that suppress the lethal phenotypes of dominant negative mutant Gre factors or overproduction of wt Gre. The mutant RNAPs will be purified, and characterized by in vitro transcription assays. #3. Interactions between Gre proteins and RNA polymerase will be explored using Fe2+-induced hydroxyl radical footprinting and mapping, and specific cysteine-directed protein-protein photochemical cross-linking. #4. To obtain three-dimensional structural information, we will prepare crystals of covalently trapped quaternary complex consisting of GreA, DNA template, RNA primer, and the Thermus thermophilus core RNA polymerase and subject them to X-ray crystallographic analysis.

描述（由申请人提供）：原核成绩单裂解 大肠杆菌中发现的Grea和Greb的因素被认为具有三个 在生物学上重要的和进化保守的转录功能： 抑制延伸逮捕，促进启动子逃生和 提高转录保真度。这些功能是由 GRE因子诱导新生RNA裂解的能力 转录复合物。该项目的广泛目标是了解 作用机理以及Grea和Greb的结构功能关系。 将进行四种类型的实验。 ＃1。为了识别功能上重要的GRE因素地区，我们将 通过寡核苷酸引入的定向随机诱变单氨基酸 替换GRE蛋白中的所有残留物除外 分子内相互作用。我们还将介绍单个和多个 保守环和该区域中的氨基酸取代 通过位置诱变之前的C末端结构域。突变体会 以体内和体外的特定转录测定为特征。 ＃2。为了阐明GRE与TC的其他组件之间的相互作用，我们 将确定RNAP的Beta和Beta Brime亚基中的突变 抑制主要阴性突变GRE因子的致命表型或 WT GRE的生产过多。突变的rnap将被纯化并表征 通过体外转录测定。 ＃3。将使用GRE蛋白与RNA聚合酶之间的相互作用。 Fe2+诱导的羟基自由基足迹和映射以及特定 半胱氨酸指导的蛋白质 - 蛋白质光化学交联。 ＃4。为了获得三维结构信息，我们将准备 共价捕获的第四纪复合物的晶体由Grea，DNA组成 模板，RNA底漆和热嗜热核RNA聚合酶和 使它们接受X射线晶体学分析。