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: 2415358
• 负责人: SERGEI BORUKHOV
• 金额: $ 17.33万
• 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/2415358
• 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.

假定的脑脊髓性转录因子谷物和greb被认为 具有两个在生物学上重要的和进化保守的功能： 抑制转录逮捕和增强 转录保真度。 这些功能是通过能力来实现的 GRE因子在三元复合物中诱导新生RNA的裂解 RNA聚合酶。 该项目的广泛目标是了解 作用的分子机制和结构功能关系 大肠杆菌中的Grea和Greb。 为此，三种类型 将进行实验。 首先，识别功能很重要 GRE因子的位置，GRE A和GRE B的氨基酸残基， 根据他们已建立的3-D结构，位于蛋白质上 表面将被诱变。 那时突变蛋白将 使用特定体外转录测定法表征生化 并通过X射线分析在结构上。 第二种实验是 针对对由GRE分子的基本“斑块”的详细研究 带正电的表面暴露残基。 为此，一系列GRE 将构造突变体，该突变体具有各种大小的基本斑块，并且 最终的突变因子将在功能上分析 生化。 暗示斑块基本残基的模型 在激活RNA聚合酶中固有核酸化位点时将是 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 - 蛋白质光化学交联研究。 cys 残留物将通过定点诱变将残留物引入GRE蛋白 选定的表面暴露残基，然后是其衍生物 硫醇特异性光活性双功能试剂。 由此产生的修改 GRE蛋白将用于探测与RNA聚合酶的相互作用 转录伸长的不同阶段。