STRUCTURE/FUNCTION OF E COLI TRANSCRIPT CLEAVAGE FACTORS

大肠杆菌转录切割因子的结构/功能

• 批准号: 2701734
• 负责人: SERGEI BORUKHOV
• 金额: $ 18.01万
• 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 2001-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2701734
• 关键词: DNA directed RNA polymerase; Escherichia coli; X ray crystallography; bacterial genetics; chemical cleavage; conformation; crosslink; cysteine; enzyme mechanism; molecular site; mutant; protein structure function; site directed mutagenesis; transcription factor; translation factor

The prokaryotic transcript cleavage factor GreA and GreB are presumed to have two biologically important and evolutionarily conserved functions: the suppression of transcription arrest and the enhancement of transcription fidelity. These functions are accomplished by the ability of Gre factors to induce the cleavage of the nascent RNA in ternary complexes of RNA polymerase. The broad goal of this project is to understand the molecular mechanism of action and the structure-functional relationships of GreA and GreB in Escherichia coli. For this purpose, three types of experiments will be conducted. First, to identify functionally important localities of Gre factors, the amino acid residues of Gre A and Gre B that, according to their established 3-D structure, are located on the protein surface will be mutagenized. The mutant proteins will be then characterized biochemically using specific in vitro transcription assays and structurally by X-ray analyses. The second type of experiments are aimed at detailed studies of the basic "patches" of Gre molecules formed by positively charged surface-exposed residues. To this end, a series of Gre mutants will be constructed that carry basic patches of various sizes and the resulting mutant factors will be analyzed functionally an biochemically. A model that implicates the basic residues of the patches in activation of the intrinsic nucleolytic site in RNA polymerase will be tested. Finally, the interactions of Gre A and Gre B with RNA polymerase will be studied using protein-protein photochemical crosslinking. The Cys residues will be introduced into Gre proteins by site-directed mutagenesis of selected surface-exposed residues followed by their derivatization with thiol-specific photoactive bifunctional reagents. The resulting modified Gre proteins will be used to probe the interactions with RNA polymerase at different stages of transcription elongation.

原核转录本裂解因子 GreA 和 GreB 被认为是 具有两个生物学上重要且进化上保守的功能： 转录停滞的抑制和增强 转录保真度。 这些功能是通过以下能力来完成的 Gre 因子诱导三元复合物中新生 RNA 的裂解 RNA聚合酶。 该项目的总体目标是了解 作用的分子机制及其结构-功能关系 大肠杆菌中的 GreA 和 GreB。 为此目的，三种类型 将进行实验。 首先，确定功能上的重要性 Gre 因子的位置，Gre A 和 Gre B 的氨基酸残基， 根据其已建立的 3-D 结构，位于蛋白质上 表面将被诱变。 突变蛋白将是 使用特定的体外转录测定进行生化表征 并通过 X 射线分析进行结构分析。 第二类实验是 旨在详细研究由以下物质形成的 Gre 分子的基本“补丁” 带正电的表面暴露残留物。 为此，一系列的Gre 将构建带有各种大小的基本补丁的突变体 由此产生的突变因子将进行功能分析 生物化学上。 暗示斑块基本残留物的模型 RNA聚合酶中内在溶核位点的激活将是 已测试。 最后，Gre A 和 Gre B 与 RNA 聚合酶的相互作用 将使用蛋白质-蛋白质光化学交联进行研究。 半胱氨酸 通过定点诱变将残基引入Gre蛋白中 选定的表面暴露残基，然后用它们衍生化 硫醇特异性光活性双功能试剂。 修改后的结果 Gre 蛋白将用于探测与 RNA 聚合酶的相互作用 转录延伸的不同阶段。