The role of collisions in rescuing stalled ribosomes in bacteria

碰撞在拯救细菌中停滞的核糖体中的作用

• 批准号: 10530678
• 负责人: Allen Rowdon Buskirk
• 金额: $ 31.17万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-02-15 至 2024-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10530678
• 关键词: Antibiotics; Bacteria; Binding; Biochemical; Biological Assay; Cells; Chemicals; Codon Nucleotides; Collaborations; Complex; Consensus; Cryoelectron Microscopy; Cues; Data; Escherichia coli; Eukaryota; Event; Failure; Genetic Screening; Goals; Kinetics; Mass Spectrum Analysis; Messenger RNA; Methods; Molecular; Open Reading Frames; Output; Pathogenesis; Pathway interactions; Predisposition; Productivity; Protein Biosynthesis; Protein Synthesis Inhibition; Proteins; Qualifying; Quality Control; Reaction; Reporter; Research Personnel; Resistance; Resolution; Ribosomal RNA; Ribosomes; Role; Signal Transduction; Site; Structure; Testing; Theoretical model; Transcript; Transfer RNA; Translating; Translations; Ubiquitin; Work; Yeasts; dimer; endonuclease; experimental study; feature detection; genetic selection; genome-wide; in vivo; insight; interest; novel; nuclease; ribosome profiling; single-molecule FRET; tmRNA; transcriptome sequencing; ubiquitin-protein ligase

PROJECT SUMMARY Translating ribosomes often encounter obstacles that stop them in their tracks: the synthesis of roughly 1 out of every 250 proteins in E. coli ends in failure. Ribosome rescue factors clear stalled ribosomes from truncated or chemically damaged mRNAs, releasing the subunits so that they can be used again. The goal of this proposal is to define the molecular mechanisms by which rescue factors such as tmRNA recognize stalled ribosomes without interfering with ribosomes engaged in productive translation. For over a decade, the consensus has been that ribosome rescue factors act on truncated mRNAs. Biochemical and structural studies indicate that tmRNA selectively reacts with ribosomes where the mRNA tunnel downstream of the ribosomal A site is empty. Yet other critical features of the recognition of stalled ribosomes may have been missed. A new paradigm has emerged in eukaryotes where stalling leads to ribosome collisions and the formation of a new interface between the small subunits of collided ribosomes. This interface is recognized by an E3 ubiquitin ligase that triggers downstream quality control events on the mRNA. The interactions between the small subunits of bacterial ribosomes in crystal lattices resemble those in collided eukaryotic disomes. Moreover, theoretical models suggest that collisions play a role in lowering protein output when stalling occurs in E. coli. At present, however, there is no direct evidence that collisions promote ribosome rescue in bacteria. We have obtained new data using reporter mRNAs loaded with different ribosome densities that show that ribosome collisions are required for tmRNA to rescue ribosomes stalled in the middle of an mRNA. Furthermore, we can purify these complexes and study their composition and structure: treating cells with an antibiotic that stalls ribosomes generates collided disomes that are nuclease-resistant. Building on these key findings, in Aim 1 we describe unbiased approaches to identify factors that recognize stalled ribosomes, including mass spectrometry of nuclease-resistant disomes and genetic selections against ribosome rescue. In Aim 2, we will use ribosome profiling to follow pausing, collisions, and rescue in vivo, asking how these phenomena change in the absence of tmRNA and novel rescue factors. Because collisions are difficult to detect in ensemble assays, we will develop single-molecule FRET methods to observe collisions and their effects on the binding kinetics of ribosome rescue factors. In addition, we will determine the structure of bacterial collided disomes (and associated factors of interest). Together, these studies will provide a comprehensive view of the role of collisions in ribosome rescue in bacteria.

项目总结 翻译核糖体经常会遇到阻碍它们前进的障碍：合成 在大肠杆菌中，大约每250种蛋白质中就有一种以失败告终。核糖体救援因素明显停滞不前 核糖体从被截断或化学损伤的mRNA中释放亚基，从而使它们能够 再次使用。这项提案的目标是定义救援因子的分子机制 例如tmRNA识别停滞核糖体而不干扰参与生产的核糖体 翻译。十多年来，人们的共识是核糖体拯救因子作用于被截断的 MRNAs。生化和结构研究表明，tmna选择性地与核糖体反应，在 核糖体A位点下游的mRNA隧道是空的。但它的其他关键特性 对停滞不前的核糖体的识别可能被遗漏了。在真核生物中出现了一种新的范式 其中失速导致核糖体碰撞并在小分子之间形成新的界面 相互碰撞的核糖体的亚基。该界面由E3泛素连接酶识别，该连接酶可触发 下游质控事件对信使核糖核酸的影响。细菌小亚基之间的相互作用 晶格中的核糖体类似于碰撞的真核双体中的核糖体。此外，理论模型 这表明，在大肠杆菌中发生失速时，碰撞在降低蛋白质产量方面发挥了作用。目前， 然而，没有直接证据表明碰撞促进了细菌中核糖体的拯救。我们有 使用负载不同核糖体密度的报告mRNA获得的新数据表明 TmRNA需要核糖体碰撞来挽救停滞在mRNA中间的核糖体。 此外，我们可以提纯这些络合物并研究它们的组成和结构：用 一种延缓核糖体的抗生素会产生耐核酸酶的碰撞二体。在基础上建设 这些关键的发现，在目标1中，我们描述了识别停滞因素的无偏见方法 核糖体，包括核糖核酸酶抗性二体的质谱学和遗传选择 核糖体拯救。在目标2中，我们将使用核糖体图谱来跟踪体内的停顿、碰撞和救援， 问这些现象在缺乏tmRNA和新的救援因子的情况下会发生什么变化。因为 在系综分析中很难检测到碰撞，我们将开发单分子FRET方法来 观察碰撞及其对核糖体救援因子结合动力学的影响。此外，我们还将 确定细菌碰撞的二体的结构(以及相关的感兴趣的因素)。加在一起，这些 研究将对碰撞在细菌核糖体拯救中的作用提供一个全面的视角。