Structure & Function of Prokaryotic Transcript Cleavage

描述（申请人提供）：原核转录物切割 在大肠杆菌中鉴定的因子GreA和GreB被认为具有三个 生物学上重要的和进化上保守的转录功能： 抑制延伸停滞，促进启动子逃逸，和 增强转录保真度。这些功能由 Gre因子诱导三元组中新生RNA切割的能力 转录复合体本项目的主要目标是了解 GreA和GreB的作用机制和构效关系。 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 通过寡核苷酸定向随机诱变引入单个氨基酸 除了那些参与Gre蛋白的残基之外， 分子内的相互作用。我们还将介绍单一和多个 保守环中的氨基酸取代和区域中的氨基酸取代 通过定点诱变在C-末端结构域之前。变种人会 通过体内和体外的特异性转录测定来表征。 #2.为了阐明Gre和TC的其他组分之间的相互作用，我们 将识别RNAP的β和β主要亚基中的突变， 抑制显性阴性突变体Gre因子的致死表型，或 过量生产wt Gre.将对突变RNAP进行纯化和表征， 通过体外转录测定。 #3. Gre蛋白和RNA聚合酶之间的相互作用将使用 Fe 2+诱导的羟基自由基足迹和作图，以及特异性 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 共价捕获的由GreA、DNA组成的四元复合物的晶体 模板、RNA引物和嗜热栖热菌核心RNA聚合酶， 对它们进行X射线晶体学分析。

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.