Control of Transcription Termination in E. coli

描述（由申请人提供）：我们的项目是探索各种蛋白质和RNA组装形成转录活性复合物的过程。蛋白质-蛋白质和rna -蛋白质的相互作用将在一个独特的可接近的遗传和生物化学系统中进行探索，即大肠杆菌和噬菌体。我们的重点是转录终止的机制和它的抑制（反终止）。我们将研究两种噬菌体蛋白，HK022 Nun终止因子和？N反终止因子。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和细胞核与四个Nus因子和RNAP形成复合体。Nus因子在抑制核糖体RNA操纵子的转录终止中也很活跃。该项目利用了我们最近分离的菌株，可以支持NusA， NusG或NusB的缺失。我们还建议分离一株携带转录不活跃的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。为了将结构和功能联系起来，我们将探讨个别领域。这些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。这种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。

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.