STRUCTURE/FUNCTION OF E COLI TRANSCRIPT CLEAVAGE FACTORS

据推测，原核转录物切割因子GreA和GreB 有两个重要的生物学和进化上保守的功能： 抑制转录停滞和增强 转录保真度 这些功能是通过以下能力来实现的： 诱导三元复合物中新生RNA裂解的Gre因子 RNA聚合酶。 本项目的主要目标是了解 分子作用机制和结构-功能关系 大肠杆菌中的GreA和GreB。 为此，三种类型的 将进行实验。 第一，确定功能重要性 Gre因子的位置，Gre A和Gre B的氨基酸残基， 根据它们已建立的三维结构， 表面将被诱变。 然后突变蛋白质 使用特异性体外转录测定进行生物化学表征 和结构上的差异。 第二类实验是 目的是详细研究基本的“补丁”的Gre分子形成的， 带正电荷的表面暴露残留物。 为此，一系列 将构建携带各种大小的基本斑块的突变体， 将对所得到的突变因子进行功能分析， 生物化学 一个模型，涉及基本残留的补丁 在RNA聚合酶的内在溶核位点的激活中， 测试. 最后，研究了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。 的Cys 通过定点突变将残基引入Gre蛋白中 选定的表面暴露的残基，然后用 巯基特异性光活性双功能试剂。 所得的修饰 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.