Structure & Function of Prokaryotic Transcript Cleavage

描述(申请人提供)：原核转录本的切割 在大肠杆菌中发现的greA和greb因子被认为有三个 生物学上重要的和进化上保守的转录功能： 抑制伸长抑制，促进启动子逃逸，以及 增强了转录的保真度。这些功能由 GRE因子诱导三元系中新生RNA裂解的能力 转录复合体。本项目的总体目标是了解 GREA和GREB的作用机制及其构效关系。 将进行四种类型的实验。 #1.为了确定GRE因子的重要功能位置，我们将 寡核苷酸定向随机诱变引入单一氨基酸 GRE蛋白中所有残基的替代，但与 分子内相互作用。我们还将推出单项和多项 保守环和区域中的氨基酸立即替换 通过定点突变先于C-末端结构域。变种人会 通过体内和体外的特异性转录实验进行鉴定。 #2.为了阐明GRE和TC的其他组成部分之间的相互作用，我们 将确定RNAP的Beta和Beta主亚单位的突变 抑制显性负性突变体Gre因子的致死表型 Wt Gre生产过剩。突变的RNAP将被纯化，并对其进行鉴定 通过体外转录实验。 #3.GRE蛋白质和RNA聚合酶之间的相互作用将使用以下方法进行探索 Fe2+诱导的羟基自由基足迹和图谱，以及特异性 半胱氨酸诱导的蛋白质-蛋白质光化学交联。 #4.为了获取三维结构信息，我们将准备 由GREA、DNA组成的共价捕获四元络合物的晶体 模板、RNA引物和嗜热嗜热菌核心RNA聚合酶和 对它们进行X射线晶体分析。

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.