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.

项目摘要 翻译核糖体经常遇到阻止它们在轨道上的障碍： 大肠杆菌中每250种蛋白质中大约有1个以失败结束。核糖体救援因素明确停滞不前 来自截短或化学受损的mRNA的核糖体释放亚基，以便它们可以是 再次使用。该提案的目的是定义救援因素的分子机制 例如TMRNA识别失速的核糖体，而不会干扰从事生产力的核糖体 翻译。十多年来，共识一直是核糖体救援因素对截断 mrnas。生化和结构研究表明，TMRNA选择性地与核糖体反应 核糖体下游的mRNA隧道位点是空的。还有其他关键特征 识别失速核糖体的识别可能已经错过。真核生物已经出现了一个新的范式 停滞导致核糖体碰撞的地方以及小之间的新界面形成 碰撞核糖体的亚基。该界面由触发的E3泛素连接酶识别 mRNA上的下游质量控制事件。细菌小亚基之间的相互作用 晶格中的核糖体类似于碰撞真核疾病的核糖体。而且，理论模型 表明，当大肠杆菌中发生失速时，碰撞在降低蛋白质输出中起作用。现在， 但是，没有直接的证据表明碰撞会促进细菌中的核糖体营救。我们有 使用载荷的记者mRNA获得了新数据 核糖体碰撞是TMRNA需要挽救停滞在mRNA中间的核糖体。 此外，我们可以净化这些复合物并研究它们的组成和结构：用 失速核糖体的一种抗生素会产生抗核酸酶的碰撞疾病。建立 这些关键发现，在AIM 1中，我们描述了识别识别停滞的因素的公正方法 核糖体，包括抗核酸酶抗性疾病的质谱和遗传选择 核糖体救援。在AIM 2中，我们将使用核糖体分析跟随暂停，碰撞和在体内营救， 询问这些现象在没有TMRNA和新型救援因素的情况下如何变化。因为 在合奏测定中很难检测到碰撞，我们将开发单分子fret方法 观察碰撞及其对核糖体救援因素的结合动力学的影响。此外，我们将 确定细菌碰撞疾病（以及相关因素）的结构。在一起，这些 研究将对碰撞在细菌中核糖体救援中的作用进行全面的看法。