Control of Transcription Termination in E. coli

描述（由申请人提供）：我们的项目是探索组装各种蛋白质和RNA以形成转录活性复合物的过程。蛋白质-蛋白质和RNA-蛋白质相互作用将在一个系统中进行探索，该系统是唯一可接近的遗传和生物化学，即E。大肠杆菌和大肠杆菌噬菌体。 我们的重点是转录终止及其抑制（antitermination）的机制。我们将研究两个噬菌体蛋白，HK 022努恩终止因子和？N抗终止因子。两者都利用宿主蛋白NusA、NusB、NusE（S10）和NusG来支持它们的反应。修女和？N绑定？？RNA与四个Nus因子和RNAP形成复合物。Nus因子在抑制核糖体RNA操纵子中的转录终止中也是活性的。 该项目利用了我们最近分离的可以支持NusA，NusG或NusB缺失的菌株。我们还提出分离具有转录失活的NusE的菌株。 根据Nus因子和噬菌体蛋白的新结构，我们将产生可能影响其转录活性的突变蛋白。我们将询问各个域，以便将结构和功能联系起来。突变体将在体内和各种体外试验中进行测试，包括纯化的转录系统、RNA结合试验和结构分析。 该原核系统和真核转录系统之间的基本相似性意味着这里获得的结果将具有广泛的生物学应用。 公共卫生相关性：转录终止的调节在原核和真核生物中都很重要。本项目主要研究E.大肠杆菌是染色体的完整性和它如何涉及核糖体释放所必需的。

DESCRIPTION (provided by applicant): Our project is to probe the processes that assemble various proteins and RNA to form a transcriptionally active complex. The protein-protein and RNA-protein interactions will be explored in a system that is uniquely approachable genetically and biochemically, namely E. coli and coliphage. We are focused on the mechanism of transcription termination and its suppression (antitermination). We will study two phage proteins, HK022 Nun termination factor and ?N antitermination factor. Both utilize host proteins, NusA, NusB, NusE (S10) and NusG to support their reactions. Nun and ?N bind ??RNA and nucleate a complex with the four Nus Factors and RNAP. The Nus factors are also active in suppressing transcription termination in ribosomal RNA operons. The project takes advantage of our recent isolation of strains that can support deletions in NusA, NusG, or NusB. We also propose to isolate a strain bearing a transcriptionally-inactive NusE. Informed by new structures of the Nus factors and the phage proteins, we will generate mutant proteins likely to affect their transcriptional activity. We shall interrogate individual domains in order to relate structure and function. The mutants will be tested in vivo and in a variety of in vitro assays, including purified transcription systems, RNA binding tests and structural analyses. The fundamental similarity between this prokaryotic system and eukaryotic transcription systems imply that the results obtained here will have broad biological application. PUBLIC HEALTH RELEVANCE: Regulation of transcription termination is important in both prokaryotic and eukaryotic organisms. This project focuses on how transcription termination in E. coli is essential for chromosome integrity and how it relates to ribosome release.