Reading frame maintenance by the ribosome during stalling

停顿期间核糖体的阅读框维护

• 批准号: 10181827
• 负责人: Hani Zaher
• 金额: $ 31.5万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-04-27 至 2025-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10181827
• 关键词: Affect; Alkylation; Amino Acids; Amino Acyl Transfer RNA; Binding; Biochemical; Biological; Cell Survival; Cells; Codon Nucleotides; Complex; Conflict (Psychology); Cryoelectron Microscopy; DNA-Directed DNA Polymerase; DNA-Directed RNA Polymerase; Data; Disease; Elements; Ensure; Eukaryota; Event; Failure; Genetic Transcription; Genomics; Goals; Lead; Life; Maintenance; Mediating; Messenger RNA; Molecular Conformation; Movement; Nature; Nucleotides; Peptides; Phase; Polyribosomes; Prevention; Process; Property; Protein Biosynthesis; Proteins; Quality Control; RNA; RNA Sequences; Reading; Reading Frames; Recovery; Regulation; Reporter; Reporting; Resolution; Resources; Ribosomal Proteins; Ribosomes; Role; Starvation; Stress; Structure; Study models; Testing; Thinness; Time; Transcript; Transfer RNA; Translation Initiation; Translations; Triplet Multiple Birth; Ubiquitination; Ursidae Family; Work; biophysical techniques; computer studies; density; experience; experimental study; fitness; genetic information; genetic resource; insight; interdisciplinary approach; interest; novel; oxidation; prevent; protein aminoacid sequence; proteostasis; recruit; response; ribosome profiling; transcriptome; translation factor; ubiquitin ligase; ubiquitin-protein ligase

PROJECT SUMMARY/ABSTRACT In all domains of life, decoding of the genetic information into peptides is accomplished by the ribosome, which reads the messenger RNA (mRNA) three nucleotides at a time. Following careful selection of the aminoacyl-tRNA that matches this triplet codon, the ribosome must precisely move to reading the next codon. Precise translocation is not an easy task given the multiple coordinated movements of the mRNA, tRNA and the ribosomal subunits that must occur. Failure to do so results in so-called frameshifting errors, which are detrimental to proteostasis as they result in errant protein products that bear no resemblance to the encoded ones. Notably, much of what we know about reading-frame maintenance comes from studies on programmed or “intentional” frameshifting. These studies revealed that sequence and structural features of the mRNA and its interaction with elements of the ribosome, translation factors and the tRNA contribute to these events. Although many of these elements are unique to each mRNA, almost all frameshifting events rely on ribosome stalling. Cellular response to stalls has been almost exclusively in the context of quality control and ribosome rescue. In particular, stalls are recognized by ubiquitin ligases when they cause ribosome collisions. In principle, colliding ribosomes can also provide structural impediments required for frameshifting; indeed, we recently showed that collisions can lead to efficient +1 frameshifting, suggesting that cells must have evolved factors to maintain reading frame during translation stalls. As ribosomes appear to stall frequently under stress, these mechanisms are more than likely to become critical for cell survival and recovery. This proposal is focused on one recently identified mechanism that involves the highly conserved multi-protein bridging factor (Mbf1). Our preliminary studies suggest that the factor prevents collision-mediated +1 frameshifting. This, together with a preliminary cyoEM structure of a Mbf1-bound ribosome, forms the basis of our major hypothesis that Mbf1 recognizes collided ribosomes to prevent them from altering the reading frame of the leading one. We will test this hypothesis through three aims. In the first one, we will assess how altering ribosome density and mRNA-sequence and -structural features modulate the function of Mbf1 in an effort to establish a relationship between ribosome collisions and frameshifting. In the second aim, using modified ribosome-profiling approaches to assess frameshifting transcriptome-wide, we will dissect the role of Mbf1 in preventing frameshifting occurring at stochastic collisions as well as those experienced under stress. In the third aim, the mechanism of Mbf1 recruitment to stalled ribosomes will be studied using a battery of biochemical and biophysical approaches. We are most interested in investigating how the factor alters the function and the structure of the translation machinery. Collectively, our interdisciplinary approach builds and expands on established expertise and resources, which we plan to use to uncover important details about how conserved factors are recruited to ribosomes to modulate their function under stress.

项目总结/摘要 在生命的所有领域，将遗传信息解码成肽是由核糖体完成的， 每次读取三个核苷酸的信使RNA（mRNA）。经过精心挑选， 当核糖体需要与这个三联体密码子匹配的氨酰-tRNA时，核糖体必须精确地移动到阅读下一个密码子。 考虑到mRNA、tRNA和RNA的多重协调运动， 核糖体亚基必须存在。否则会导致所谓的移帧错误， 对蛋白质稳态有害，因为它们导致错误的蛋白质产物， 一个值得注意的是，我们对阅读框架维持的了解大部分来自对编程的研究。 或“故意”移框。这些研究揭示了mRNA的序列和结构特征， 它与核糖体元件、翻译因子和tRNA的相互作用促成了这些事件。 尽管这些元件中有许多是每个mRNA所特有的，但几乎所有的移码事件都依赖于核糖体。 拖延时间细胞对失速的反应几乎完全是在质量控制和核糖体的背景下进行的 救援特别是，当泛素连接酶引起核糖体碰撞时，失速被泛素连接酶识别。在 原则上，碰撞的核糖体也可以提供移码所需的结构障碍;事实上，我们 最近表明，碰撞可以导致有效的+1移码，这表明细胞必须进化 在转换暂停期间保持阅读帧的因素。由于核糖体在低温下似乎经常停滞， 在压力下，这些机制更有可能成为细胞存活和恢复的关键。这项建议 是集中在一个最近确定的机制，涉及高度保守的多蛋白质桥接 因子（Mbf 1）。我们的初步研究表明，该因子阻止了碰撞介导的+1移码。 这与Mbf 1结合的核糖体的初步cyoEM结构一起，形成了我们的主要研究基础。 假设Mbf 1识别碰撞的核糖体，以防止它们改变核糖体的阅读框架。 领导一我们将通过三个目标来检验这一假设。在第一个，我们将评估如何改变 核糖体密度和mRNA序列和结构特征调节Mbf 1的功能， 建立核糖体碰撞和移码之间的关系。在第二个目标中，使用修改的 核糖体分析方法来评估转录组范围内的移码，我们将剖析Mbf 1的作用， 在防止随机碰撞以及在压力下经历的帧移位中发生。在 第三个目标，Mbf 1招募到停滞核糖体的机制将使用一组 生物化学和生物物理方法。我们最感兴趣的是研究这个因素如何改变 翻译机器的功能和结构。总的来说，我们的跨学科方法建立和 扩展了现有的专业知识和资源，我们计划使用这些知识和资源来揭示有关 保守因子如何被募集到核糖体中以调节它们在压力下的功能。