The structural dynamics of ribosomal frameshifting and ribosome rescue

核糖体移码和核糖体拯救的结构动力学

基本信息

批准号：
10377976
负责人：
Ruben L Gonzalez
金额：
$ 39.46万
依托单位：
COLUMBIA UNIV NEW YORK MORNINGSIDE
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-05-01 至 2024-02-29
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10377976
关键词：
Address Amino Acids Antibiotics Bacteria Bacterial Model Binding Biochemical Biological Clinical Codon Nucleotides Collaborations Complement Cryoelectron Microscopy Data Development Disease Electron Microscopy Eukaryota Event Fluorescence Frameshift Mutation Gene Expression Genome Goals Health Human In Vitro Investigation Kinetics Label Laboratories Letters Life Link Malignant Neoplasms Mediating Messenger RNA Modeling Molecular Conformation Molecular Target Motivation Nucleotides Organism Play Positioning Attribute Process Production Property Protein Biosynthesis Proteins Reporting Resolution Rest Ribosomal Frameshifting Ribosomal Proteins Ribosomes Role Saccharomyces cerevisiae Series Signal Transduction Specific qualifier value Structure System Techniques Technology Testing Time Transfer RNA Translations Viral Cancer Virus Diseases Work Yeasts base cryogenics experimental study flexibility fluorescence imaging fluorophore human disease next generation polypeptide public health relevance reconstitution single molecule single-molecule FRET small molecule small molecule therapeutics

项目摘要

PROJECT SUMMARY Translation of messenger RNAs (mRNAs) into proteins by the ribosome and the rest of the translation machinery (TM) is a fundamental step in gene expression that is central to life. Because the bacterial TM is a proven target for the development of new antibiotics and because many human diseases have been causally linked to dysregulation of translation, the mechanisms of translation and translational control in bacteria and eukaryotes remain under intense investigation. Over the past two decades, structural studies have revealed the large-scale structural rearrangements the TM undergoes during protein synthesis. Unfortunately, the size, complexity, and conformational flexibility of the TM have greatly impeded studies of these dynamics, significantly limiting our understanding of how these dynamics contribute to the mechanisms of translation and translational control. Nonetheless, using a combination of single-moleucle fluorescence and structural techniques, we and others have been able to characterize the dynamics of the core steps of translation by the bacterial TM. Despite these accomplishments, critical gaps in our understanding remain regarding whether and how the dynamics of these core steps are modulated as part of biomedically important translational control strategies. To fill these gaps, in the first aim of this application, we propose to use a combination of single- molecule fluorescence, structural, and biochemical approaches to investigate how the dynamics of the bacterial TM are modulated in order to drive ribosomal frameshifting. Frameshifting is a translational control strategy in which the TM slips backward or forward by one or more nucleotides on the mRNA to either correct an insertion or deletion ‘frameshift’ mutation that would otherwise result in production of an aberrant or truncated protein or to drive the synthesis of more than one protein product from a single mRNA. These experiments promise to reveal the still-elusive mechanism(s) that underlie frameshifting. In the second aim, we will use analogous approaches to investigate how ribosome rescue factors modulate the dynamics of the bacterial TM as part of the mechanisms through which they recognize and rescue ribosomes that have become translationally compromised. These studies will provide structure-based mechanistic models of bacterial ribosome rescue systems that can be exploited in the development of new antibiotics. In the third aim, we will extend our combination of single-molecule fluorescence and structural techniques to a yeast translation system, enabling us to investigate eukaryotic-specific aspects of the core steps of translation, frameshifting, and ribosome rescue. The results of these studies will reveal the mechanisms that drive and regulate translation in eukaryotes, providing a framework for investigating the role of translational control in human health and disease.

项目概要通过核糖体将信使 RNA (mRNA) 翻译成蛋白质以及其余翻译过程机器（TM）是基因表达的基本步骤，是生命的核心，因为细菌TM是一个基因表达过程。已被证明是开发新抗生素的目标，并且因为许多人类疾病已被证实是因果关系与翻译失调、细菌的翻译机制和翻译控制有关在过去的二十年里，结构研究揭示了真核生物的存在。不幸的是，TM 在蛋白质合成过程中经历了大规模的结构重排。 TM 的复杂性和构象灵活性极大地阻碍了对这些动力学的研究，极大地限制了我们对这些动态如何促进翻译机制的理解尽管如此，使用单分子荧光和结构的组合。技术，我们和其他人已经能够通过尽管取得了这些成就，但我们对于是否和细菌的理解仍然存在重大差距。作为生物医学重要的翻译控制的一部分，如何调节这些核心步骤的动态为了填补这些空白，在本应用程序的第一个目标中，我们建议使用单一策略的组合。分子荧光、结构和生化方法来研究细菌 TM 被调节以驱动核糖体移码移码是一种翻译控制。 TM 在 mRNA 上向后或向前滑动一个或多个核苷酸以纠正错误的策略插入或删除“移码”突变，否则会导致产生异常或截短的蛋白质或驱动单个 mRNA 合成多种蛋白质产物。实验有望揭示移码背后仍然难以捉摸的机制。在第二个目标中，我们。将使用类似的方法来研究核糖体救援因子如何调节细菌TM作为其识别和拯救核糖体机制的一部分这些研究将提供基于结构的力学模型。可用于开发新抗生素的细菌核糖体救援系统。我们将把单分子荧光和结构技术的结合扩展到酵母翻译系统，使我们能够研究翻译、移码、这些研究的结果将揭示驱动和调节的机制。真核生物翻译，为研究翻译控制在人类中的作用提供了一个框架健康和疾病。