Mechanisms for Stress-Induced Transcriptional Reprogramming via Anti-Adaptors

项目摘要 可分离的启动子识别亚基RpoS（也称为σs）是转录调控因子的主要转录调控因子。 γ-变形菌的一般应激反应，并在许多病原体的毒力中起关键作用， 包括人类、植物和动物病原体。在某些条件下，例如在从 与稳定期成对数或在存在应激信号的情况下，RpoS重定向核心RNA， 聚合酶机器与启动子的子集连接以重编程转录。然而，细胞内RpoS水平 不稳定-它们在活跃分裂的细胞中很低，并且在进入静止期后大幅增加。 阶段或遇到压力时。为了实现适当的监管，对RpoS水平进行严格控制， 通过ATP依赖性ClpXP机器在RpoS蛋白水解水平上发生的主要调节点。 我们的中心重点是了解ClpXP对RpoS蛋白水解的机制，以及ClpXP对RpoS蛋白水解的调控。 一个新兴的蛋白质家族，统称为抗适配器。为了降级，RpoS被呈现为 ClpXP通过一个独特的，高度特异性的适配器称为RssB，它起催化作用，而不会被降解。在 反过来，RssB本身通过与应激特异性抗适应因子的相互作用来调节。我们的工作将集中在 三抗衔接子结构和功能：由氧化应激和DNA损伤诱导的CD 3D，CD 3 M （由镁饥饿诱导的）和磷酸盐饥饿诱导的）。这些反适应者没有 序列同源性，并且与已知结构的蛋白质只有弱同源性，这表明结构生物学 旨在破译RssB识别的潜在机制的努力。我们将确定 使用X射线晶体学，分离并与RssB结合的RssD、RssM和RssP。这将使我们能够 以原子分辨率精确定位对抗适配器/RssB相互作用重要的残基，以及对 抗衔接子本身通过寡聚化。我们将补充这些结构研究与分子 遗传学，显微镜和功能测定蛋白质-蛋白质相互作用和RpoS降解，这将 使我们能够将体外行为与体内观察相关联。我们还将确定RPOS的结构- RssB-ClpXP组装使用电子冷冻显微镜的尖端方法，这将使我们能够 了解RpoS和RssB构象动力学的核心，这种范式模式的调节 细菌中的蛋白质水解。总的来说，这项工作不仅会带来基本的，机械的见解，而且会 开辟了一条新的抗菌药物，可以直接靶向ClpXP，或适配器/抗- 适配器接口。据报道，RpoS调节子占大肠杆菌的10 RpoS本身在细菌的持久性、宿主-病原体相互作用和生物膜中起着重要作用 形成，这是80%的感染的基础。

PROJECT SUMMARY The dissociable promoter recognition subunit RpoS (also known as σs) is the master transcriptional regulator of the general stress response in γ-proteobacteria, and plays key roles in the virulence of many pathogens, including human, plant and animal pathogens. Under certain conditions, such as at the transition from the logarithmic to the stationary phase or in the presence of stress signals, RpoS redirects the core RNA polymerase machinery to a subset of promoters to reprogram transcription. However, intracellular RpoS levels are not steady – they are low in actively dividing cells, and substantially increased upon entering the stationary phase or upon encountering stress. To achieve proper regulation, there is tight control over RpoS levels, with the major point of regulation occurring at the level of RpoS proteolysis by the ATP-dependent ClpXP machine. Our central focus is to understand the mechanisms of RpoS proteolysis by ClpXP as well as its regulation by an emerging family of proteins collectively called anti-adaptors. In order to be degraded, RpoS is presented to ClpXP by a unique, highly specific adaptor called RssB, which acts catalytically, without being degraded. In turn, RssB itself is regulated by interactions with stress-specific anti-adaptors. Our work will focus on the structure and function of three-anti-adaptors: IraD (induced by oxidative stress and DNA damage), IraM (induced by magnesium starvation) and IraP (induced by phosphate starvation). These anti-adaptors share no sequence homology, and only weak homology with protein of known structure, warranting a structural biology effort aimed at deciphering the underlying mechanisms of RssB recognition. We will determine the structures of IraD, IraM and IraP, both in isolation and bound to RssB using X-ray crystallography. This will allow us to pinpoint, at atomic resolution, residues important for anti-adaptor/RssB interactions, and also regulation of the anti-adaptors themselves by oligomerization. We will complement these structural studies with molecular genetics, microscopy and functional assays for protein-protein interactions and RpoS degradation, which will allow us to correlate in vitro behavior with in vivo observations. We will also determine structures of a RpoS- RssB-ClpXP assembly using cutting-edge methods in electron cryo-microscopy, which will allow us to understand the RpoS and RssB conformational dynamics at the core of this paradigmatic mode of regulated proteolysis in bacteria. Overall, this work will not only bring fundamental, mechanistic insights, but will also open the way to the development of novel antibacterials that could target ClpXP directly, or, adaptor/anti- adaptor interfaces. The RpoS regulon has been reported to comprise up to 10% of the Escherichia coli genome, and RpoS itself plays important roles in bacterial persistence, host-pathogen interactions and biofilm formation, which underlie 80% of all infections.