Transcription in E. coli: Structural Basis of Nus-Factor-Dependent Regulation

转录终止和抗终止之间的竞争构成了基因调控的关键机制，例如λ噬菌体裂解生长期间或大肠杆菌核糖体RNA（rrn）转录期间的范式进行性抗终止以及由噬菌体HK 022诱发的提前终止。这些过程依赖于RNA信号元件（N-利用，nut，位点），噬菌体蛋白N（λ）或Nun（HK 022）和四种宿主N-利用物质（NusA，NusB，NusE [相当于核糖体蛋白S10]和NusG），它们在RNA聚合酶（RNAP）表面组装复杂的核糖核蛋白复合物。在这里，我建议调查终止/antitermination在上述系统的分子机制的综合方法，建立在我自己的小组（X射线晶体学）和保罗Rösch（大学拜罗伊特，NMR光谱）的合作小组的结构生化专业知识。具体而言，我们打算研究NusB协助SIO的多种功能的机制;阐明NusB-S10复合物与boxA RNA结合的模式;研究NusG与NusB-S10-boxA RNA复合物、RNA聚合酶和转录终止因子Rho的功能相互作用;并开发组装整个转录终止和抗终止复合物的方法，用于结构和功能研究。除了阐明转录和翻译的基本调控原则外，拟议工作的结果将为开发抗菌物质提供新的平台。

Competition between transcription termination and antitermination constitutes a key mechanism of gene regulation, as exemplified by the paradigmatic processive antitermination during lytic growth of phage λ or during ribosomal RNA (rrn) transcription in Escherichia coli and by the premature termination evoked by phage HK022. These processes depend on an RNA signal element (the N-utilization, nut, site), phage proteins N (λ) or Nun (HK022) and four host N-utilization substances (NusA, NusB, NusE [equivalent to ribosomal protein S10] and NusG), which assemble elaborate ribonudeoprotein complexes on the surface of RNA polymerase (RNAP). Here, I propose to investigate the molecular mechanisms underlying termination/antitermination in the above systems by an integrative approach, building on structural biochemical expertise of my own group (X-ray crystallography) and of the collaborating group of Paul Rösch (Universität Bayreuth, NMR spectroscopy). Specifically, we intend to investigate the mechanisms by which NusB assists the diverse functions of SIO; to elucidate the mode of boxA RNA binding by the NusB-S10 complex; to investigate the functional interplay of NusG with the NusB-S10-boxA RNA complex, with RNA polymerase and with transcription termination factor Rho; and to develop methods to assemble entire transcription termination and antitermination complexes for structural and functional studies. Apart from shedding light onto fundamental regulatory principles of transcription and translation, the outcome of the proposed work will provide novel platforms for the development of antimicrobial substances.