Control of Transcription Termination in E.coli

描述(申请人提供)：在转录终止水平上的基因表达调节在自然界中是普遍存在的。然而，终止反应的确切机制，即使在细菌中，也只有部分了解。噬菌体HK022 Nun蛋白与新生噬菌体lambda转录本结合于特定位点(NUT)，在体外诱导转录停止，在体内终止转录。Nun和RNA聚合酶与宿主因子NusA、NusB、NusE和NusG的相互作用参与了这一反应。宿主MFD蛋白在体外和体内释放Nun滞留的RNA聚合酶。我们将探索Nun与类似于Nusa Nun分子内接触的宿主因子相互作用的可能性，以及Nun与宿主蛋白之间的相互作用将由化学和遗传方法确定。半胱氨酸取代的NUN将与交联剂衍生化，并与RNA聚合酶和NUS因子反应。交联物的位置将通过质谱学确定。半胱氨酸取代的NUN也将被用来通过FRET分析确定NUN在NUN-RNA聚合酶复合体中的位置。NUN突变体将根据它们与RNA聚合酶和NusA的相互作用来表征。Nun和相关的lambda蛋白N与坚果和lambda Pl转录本中的RNaseIII靶点的结合将在体内和体外进行研究。将研究NUN对大肠杆菌毒性的基础，并测试NUN识别除坚果以外的序列的可能性。

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.