STRUCTURE/FUNCTION OF E COLI TRANSCRIPT CLEAVAGE FACTORS

原核转录产物切割因子GreA和Greb被推测为 有两个生物学上重要的和进化上保守的功能： 抑制转录抑制和增强转录抑制 转录保真度。这些功能是通过以下能力实现的 GRE因子诱导三元复合体中新生RNA的裂解 RNA聚合酶。本项目的总体目标是了解 黄曲霉毒素的分子作用机制及其构效关系 Grea和Greb在大肠杆菌中的表达。为此，有三种类型的 将进行实验。第一，确定重要的功能 Gre因子的位置，Gre A和Gre B的氨基酸残基， 根据它们建立的三维结构，都位于蛋白质上 表面将被诱变。突变的蛋白质将会是 用特定的体外转录实验进行生化表征 并通过X射线分析对其结构进行分析。第二类实验是 旨在详细研究GRE分子形成的基本斑块 带正电的表面暴露残留物。为此，一系列GRE 将构建携带各种大小和基本斑块的突变体 由此产生的突变因子将在功能上和 生化方面的。一个包含斑块的基本残基的模型 在激活RNA聚合酶中的固有核溶解位点时 测试过。最后，Gre A和Gre B与RNA聚合酶的相互作用 将利用蛋白质-蛋白质光化学交联法进行研究。赛斯家族 通过定点突变将残基引入GRE蛋白 选定的表面暴露的残留物，然后用 硫醇专一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 转录延伸的不同阶段。

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.