The role of collisions in rescuing stalled ribosomes in bacteria

项目摘要 翻译核糖体经常会遇到阻碍，使它们停止在它们的轨道上： 大约每250个蛋白质中就有一个coli以失败告终。核糖体拯救因子明显停滞 核糖体从截断或化学损伤的mRNA，释放亚基，使它们可以 再次使用。这项建议的目的是确定拯救因子的分子机制， 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截短的 mRNA。生物化学和结构研究表明，tmRNA选择性地与核糖体反应， 核糖体A位点下游的mRNA通道是空的。然而， 可能错过了对停滞核糖体的识别。在真核生物中出现了一种新的模式 在那里停滞导致核糖体碰撞，并在小分子之间形成新的界面， 碰撞核糖体的亚基。该界面被E3泛素连接酶识别， mRNA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的真核二体中的核糖体。此外，理论模型 表明当在E.杆菌目前， 然而，没有直接证据表明碰撞促进细菌中核糖体拯救。我们有 使用装载有不同核糖体密度的报告mRNA获得了新的数据， tmRNA需要核糖体碰撞来拯救停滞在mRNA中间的核糖体。 此外，我们可以纯化这些复合物并研究它们的组成和结构： 一种阻止核糖体的抗生素会产生抗核酸酶的碰撞二体。基础上 这些关键发现，在目标1中，我们描述了无偏见的方法，以确定因素，承认停滞 核糖体，包括核酸酶抗性二体的质谱分析和基因选择， 核糖体拯救在目标2中，我们将使用核糖体分析来跟踪体内的暂停、碰撞和拯救， 询问这些现象在缺乏tmRNA和新的拯救因子的情况下如何改变。因为 碰撞是很难检测在合奏测定，我们将开发单分子FRET方法， 观察碰撞及其对核糖体拯救因子结合动力学的影响。此外，我们将 确定细菌碰撞二体的结构（和相关的感兴趣的因素）。所有这些 研究将提供一个全面的看法碰撞的作用，核糖体拯救细菌。

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.