Structural dynamics underlying the multi-functionality of a global gene regulator

在所有已测序基因组的细菌物种中，超过90%的细菌表达依赖于六聚体RNA的NTPase，p在其中许多细菌中是必不可少的。长期以来，P一直被认为是转录终止的中介，它定义了例如，在大肠杆菌中20-30%的转录单位的末端。然而，最近的发现揭示了p的一种意想不到的多功能，通过p作为全球基因调节因子和表型异质性因子，增加了细菌在不利条件下生存的机会。例如，p还可以调节5‘-非翻译区的衰减，限制反义转录的程度，沉默外源基因，并通过限制R-环来保护基因组。P的多功能性显然是通过它与其他蛋白质和RNA元件的直接或间接相互作用而建立的。尽管进行了几十年的研究，但对ρ依赖的转录终止的分子机制以及p相互作用的蛋白质和RNA如何调节和多样化p的活性和功能仍知之甚少。本项目将全面介绍ρ的S与转录延伸复合体以及与其他调节延伸复合体上ρ活性的蛋白质和RNA的动态相互作用的结构基础，并揭示这些相互作用如何促进p在细胞中的许多作用。它包括捕捉和结构分析NTP驱动的多步骤过程中的中间阶段和调控情况，这些过程是由大而灵活的分子机制介导的，并同时描绘这些大分子复合体在这些过程中经历的构象和组成变化。此外，还将利用结构洞察力来指导体外和体内的功能分析，包括基因组/转录组范围的研究。通过组装精心设计、p修饰和p调制的内皮细胞，并利用最近在单粒子冷冻电子显微镜方面的突破，我们已经在实现这些雄心勃勃的目标方面取得了重大进展。预期的结果将揭示细菌基因调控的基本原理，开发的方法将为动态大分子复合体的特定阶段分析提供蓝图，以获得超出这里研究的特定系统的反应周期的原子细节的电影般的表示。

More than 90 % of all bacterial species with sequenced genomes express the hexameric RNA-dependent NTPase, p. p is essential in many of these bacteria. p is long known as a mediator of transcription termination that defines, for instance, the ends of 20-30 % of transcription units in Escherichia coli. However, recent findings have uncovered an unexpected multi-functionality of p, through which p acts as a global gene regulator and as a phenotypic heterogeneity factor that increases chances for survival of bacteria under adverse conditions. For example, p can, among others, also mediate attenuation in 5’-untranslated regions, limit the extent of antisense transcription, silence foreign genes and safeguard genomes by restricting R-loops. The multi-functionality of p is apparently established through its direct or indirect interactions with other proteins and RNA elements. Despite decades of research, the molecular mechanisms underlying ρ-dependent transcription termination and how p-interacting proteins and RNAs modulate and diversify p activity and function are poorly understood. This project will provide a comprehensive view on the structural basis of ρ’s dynamic interactions with transcription elongation complexes as well as with other proteins and RNAs that modulate ρ activity on elongation complexes, and will reveal how these interactions facilitate p’s many roles in cells. It involves capturing and structurally analyzing intermediate stages and regulatory situations along NTP-driven, multi-step processes that are mediated by large and flexible molecular machinery, and concomitantly delineating the conformational and compositional changes that these macromolecular complexes undergo during these processes. Moreover, structural insights will be exploited to guide functional analyses in vitro and in vivo, including genome/transcriptome-wide investigations. Assembling carefully designed, p-modified and p-modulating ECs and capitalizing on recent breakthroughs in single-particle cryo-electron microscopy, we have already made major progress towards these ambitious goals. Expected results will uncover fundamental principles of bacterial gene regulation, and methods developed will provide a blueprint for the stage-specific analysis of dynamic macromolecular complexes, to obtain movie-like representations in atomic detail of reaction cycles beyond the specific systems studied here.