Control of Transcription Termination in E.coli

描述（由申请人提供）：在转录终止水平的基因表达调控在自然界中是普遍存在的。然而，即使在细菌中，终止反应的确切机制也只是部分地被了解。噬菌体HK022 Nun蛋白在特定位点（坚果）结合新生噬菌体lambda转录物，诱导体外转录阻滞和体内转录终止。RNA聚合酶与宿主因子NusA、NusB、NusE和NusG之间的相互作用参与了该反应。宿主Mfd蛋白在体外和体内释放非捕获RNA聚合酶。我们将探索Nun与类似于NusA的宿主因子相互作用的可能性。Nun分子内接触以及Nun与宿主蛋白之间的相互作用将通过化学和遗传方法来定义。半胱氨酸取代的Nus将与交联剂衍生，并与RNA聚合酶和Nus因子反应。交联的位置将由质谱法确定。半胱氨酸取代的Nun也将用于通过FRET分析确定Nun在rna聚合酶复合体中的位置。尼农突变体将根据其与RNA聚合酶和NusA的相互作用来表征。将在体内和体外研究Nun和相关的lambda蛋白N与坚果和lambda pL转录物中RNaseIII靶位点的结合。研究人员将调查Nun对大肠杆菌毒性的基础，并测试其识别坚果以外序列的可能性。

DESCRIPTION (provided by applicant): Regulation of gene expression at the level of transcription termination is ubiquitous in nature. Nevertheless the precise mechanism of the termination reaction, even in bacteria, is only partially understood. Coliphage HK022 Nun protein binds nascent phage lambda transcript at specific sites (nut) and induces transcription arrest in vitro and termination in vivo. Interactions between Nun and RNA polymerase and host factors NusA, NusB, NusE and NusG are involved in this reaction. Host Mfd protein releases Nun-arrested RNA polymerase in vitro and in vivo. We will explore the possibility that Nun interacts with a host factor analogous to NusA Nun intramolecular contacts as well as interactions between Nun and host proteins will be defined by chemical and genetic approaches. Cysteine-substituted Nun will be derivatized with cross-linkers and reacted with RNA polymerase and Nus factors. The location of the cross-link will be determined by Mass Spectrometry. Cysteine-substituted Nun will also be used to determine the location of Nun in a Nun-RNA polymerase complex by FRET analysis. Nun mutants will be characterized with respect to their interactions with RNA polymerase and NusA. Binding of Nun and the related lambda protein, N, to nut and to the RNaseIII target site in the lambda pL transcript will be studied in vivo and in vitro. The basis of Nun toxicity to E. coli will be investigated and the possibility that Nun recognizes sequences other than nut will be tested.