Control of Transcription Termination in E-Coli

描述(申请人提供)：在大肠杆菌中，RNA聚合酶和前导核糖体可能通过两个结构域的NusG蛋白物理连接。这将在生化、结构和遗传水平上得到证明。我们打算分离FL NusG和70年代核糖体之间的一个复合体，并使用冷冻-EM来探索其结构。我们将筛选一些突变体，以了解它们对偶联转录和翻译的影响。我们的结果表明，关系 是配对所必需的，这将从遗传学和生物化学的角度进行探索。我们的研究表明，当翻译被抑制时，tmRNA具有使转录和翻译解偶联的功能。结构数据表明，tmRNA可以取代核糖体中的NusG；我们将在体外测试这一点。核磁共振和生化分析表明，NusA可以取代RNAP中的NusG。这将在一个纯化的转录系统中进行测试。染色体的完整性依赖于转录终止。我们将探讨这一反应的细节。最后，我们发现转录与多种DNA损伤的修复有关。转录的影响取决于DNA损伤的类型。我们将探索修复DNA双链断裂、DNA加合物和链间交联的途径，以及从DNA模板中移除紧密结合的蛋白质的途径。

DESCRIPTION (provided by applicant): RNA polymerase and the lead ribosome may be physically linked in E. coli by the two-domain NusG protein. This will be demonstrated at the biochemical, structural and genetic levels. We intend to isolate a complex between FL NusG and the 70S ribosome, and to probe its structure using cryo-EM. We will screen a number of mutants for their effects on coupling transcription and translation. Our results indicate that RelA is required for coupling and this will be explored genetically and biochemically. Our studies have implicated tmRNA as the function that uncouples transcription and translation when translation is inhibited. Structural data indicates that tmRNA can displace NusG from the ribosome; we will test this in vitro. NMR and biochemical analyses suggest that NusA can displace NusG from RNAP. This will be tested in a purified transcription system. Chromosome integrity depends on transcription termination. The details of this reaction will be explored. Finally, we find that transcription is linked to repair of a variety of DNA damages. The influence of transcription depends on the type of DNA damage. We will explore the pathways that repair DNA DSBs, DNA adducts, and interstrand crosslinks, and that can remove tightly bound protein from the DNA template.