CONTROL OF TRANSCRIPTION TERMINATION IN E COLI

大肠杆菌转录终止的控制

• 批准号: 2734549
• 负责人: MAX Elliot GOTTESMAN
• 金额: $ 45.09万
• 依托单位: COLUMBIA UNIVERSITY HEALTH SCIENCES
• 依托单位国家: 美国
• 财政年份: 1986
• 资助国家: 美国
• 起止时间: 1986-07-01 至 1999-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2734549
• 关键词: DNA directed RNA polymerase; Escherichia coli; RNase protection assay; bacterial genetics; bacteriophage lambda; gene expression; gene induction /repression; gene mutation; genetic mapping; genetic promoter element; genetic transcription; genetic translation; host organism interaction; messenger RNA; microorganism culture; molecular cloning; nucleic acid sequence; operon; regulatory gene; temperature sensitive mutant; transcription factor; transcription termination; virus protein

DESCRIPTION: This grant has supported study of both transcription and chaperone function, and now is concentrated on transcription - at this stage, mainly the mechanism of transcription termination mediated by a specific bacteriophage protein, Nun, and also the function of an essential cellular protein NusG that is (at least) an accessory to rho- dependent termination. 1) NusG affects both termination efficiency at rho-dependent terminators and the related processes of lambda N-mediated antitermination and Nun-mediated termination (see below). Nun is thought to interact with other N accessory factors, like NusA. Dr. Gottesman plans to use a Nun-resistant NusG (NusG*) to select phage and cellular suppressors that define interactions with other proteins; in one approach they will specifically mutagenize candidates like nusA, nusB, and nusE. They will try to isolate nusG ts mutations, and N-resistant nusG mutations, as well as nusG mutations that affect cellular termination. 2) Nun is a protein made by HKO22 that is like Nit binds the same RNA site (named nut) and uses the same cofactors but terminates instead of antiterminating. It works in vitro with purified components, as does N, but slightly defectively, because the RNA does not release. They will look for release, either by the obvious known candidate GreB, or by unknown cellular release factors in extracts. They will isolate release defective mutants of E. coli, under the assumption that arrest of transcription by Nun along with failure of release will induce constipation. They will investigate the structure of Nun and the Nun- RNA complex: the effect of mutants of the nut site known to affect N, RNase protection patterns, the activity of fragments of Nun, the activity of Nun carrying modification of putative critical arginine residues, and (by collaboration) NMR structure of Nun-RNA complex. They will seek a Nun with altered specificity for a mutant nut site and look for suppressors of mutations in accessory factors or RNA polymerase (NusA, NusG, rpoC). They will look for contacts by crosslinking, and characterize protein components of Nun-arrested complexes. They will characterize RNA at sites of arrest (as opposed to recognition). Finally, they will investigate a natural proteolytic cleavage, looking for the protease, and isolate E. coli mutants resistant to the activity of Nun.

描述：该赠款支持对转录和 伴侣功能，现在集中于转录 - 此 阶段，主要是由A介导的转录终止机理 特定的噬菌体蛋白，修女以及 必需的细胞蛋白NUSG（至少）是Rho-的附件 依赖终止。 1）NUSG影响两个终止效率 依赖Rho的终结器和Lambda N介导的相关过程 抗授予和修女介导的终止（见下文）。 修女是想念的 与其他N附件因子（例如NUSA）互动。 戈特斯曼博士 计划使用抗修女的NUSG（NUSG*）选择噬菌体和蜂窝 定义与其他蛋白质相互作用的抑制剂；一个 他们将专门化NUSA，NUSB等候选人的诱变 和nuse。他们将尝试隔离NUSG TS突变，并抗N- NUSG突变以及影响细胞的NUSG突变 终止。 2）修女是hko22制造的蛋白质，就像nit结合 相同的RNA位点（命名螺母）并使用相同的辅助因子，但终止 而不是抗精。 它可以在体外与纯化的组件一起工作， 就像n一样，但略有缺陷，因为RNA不会释放。 他们将寻找明显已知的候选人Greb的释放， 或通过提取物中未知的细胞释放因子。 他们会孤立 假设释放大肠杆菌的有缺陷的突变体 修女的转录以及释放失败将引起 便秘。 他们将研究修女和修女的结构 RNA复合物：已知会影响n的坚果位点突变体的影响 RNase保护模式，修女碎片的活性， 修改假定的临界精氨酸的修饰的活动 残基，（通过协作）Nun-RNA复合物的NMR结构。 他们 将寻求因突变坚果部位而具有改变特异性的修女，并查看 用于辅助因子或RNA聚合酶中突变的抑制剂 （NUSA，NUSG，RPOC）。 他们将通过交联寻找联系，然后 表征了修女避难的复合物的蛋白质成分。 他们会的 在逮捕部位（而不是认可）的RNA特征。 最后，他们将研究天然的蛋白水解裂解，看上去 对于蛋白酶和分离型大肠杆菌突变体对活性的抗性 修女。