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形成转录活性复合体的过程。蛋白质-蛋白质和RNA-蛋白质的相互作用将在一个在遗传和生物化学上唯一接近的系统中进行探索，即大肠杆菌和大肠杆菌。我们主要关注转录终止的机制及其抑制(反终止)。我们将研究两种噬菌体蛋白，HK022 Nun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来支持他们的反应。NUN和？N结合核糖核酸，并与四种NUS因子和RNAP形成复合体。NUS因子在抑制核糖体RNA操纵子的转录终止方面也是活跃的。该项目利用了我们最近分离的能够支持NusA、NusG或NusB缺失的菌株。我们还建议分离出一株携带转录不活跃神经的菌株。受NUS因子和噬菌体蛋白新结构的影响，我们将产生可能影响其转录活性的突变蛋白。我们将询问各个领域，以便将结构和功能联系起来。这些突变体将在体内和各种体外测试中进行测试，包括纯化的转录系统、RNA结合测试和结构分析。这个原核系统与真核转录系统之间的基本相似性表明，本文所获得的结果将具有广泛的生物学应用价值。 公共卫生相关性：转录终止的调节在原核生物和真核生物中都很重要。这个项目的重点是在大肠杆菌中转录终止是如何对染色体完整性至关重要的，以及它如何与核糖体释放有关。

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.