Reading frame maintenance by the ribosome during stalling

停顿期间核糖体的阅读框维护

• 批准号: 10596204
• 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/10596204
• 关键词: Affect; Alkylation; Amino Acids; Amino Acyl Transfer RNA; Binding; Biochemical; Biological; Cell Survival; Cells; Codon Nucleotides; Complex; Cryoelectron Microscopy; DNA-Directed DNA Polymerase; DNA-Directed RNA Polymerase; Data; Disease; Elements; Ensure; Eukaryota; Event; Failure; Genetic Transcription; Genomic Instability; Goals; Initiator Codon; Lead; Life; Maintenance; Mediating; Messenger RNA; Molecular Conformation; Movement; Nature; Nucleotides; Peptides; Phase; Polyribosomes; Prevention; Process; Property; Protein Biosynthesis; Proteins; Quality Control; 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; Work; biophysical techniques; computer studies; conflict resolution; 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.

项目摘要/摘要 在生命的所有领域中，将遗传信息解码为胡椒体是由核糖体完成的， 它一次读取Messenger RNA（mRNA）三个核苷酸。仔细选择 与此三重码密码子相匹配的氨基酰基-TRNA，核糖体必须精确地移至读取下一个密码子。 鉴于mRNA，tRNA和 必须发生的核糖体亚基。如果不这样做会导致所谓的架空错误，这是 对蛋白质的有害，因为它们会导致与编码不相似的错误蛋白质产物 一个。值得注意的是，我们对阅读框架维护的了解大部分来自对编程的研究 或“有意”的框架。这些研究表明mRNA和 它与核糖体元素，翻译因子和tRNA的相互作用有助于这些事件。 尽管这些元素中的许多是每个mRNA所独有的，但几乎所有的框架事件都依赖于核糖体 存储。细胞对摊位的响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，当引起核糖体碰撞时，泛素连接酶会识别摊位。 原理，碰撞核糖体还可以提供壁画所需的结构障碍；确实，我们 最近表明，碰撞可能导致有效的+1帧速率，这表明细胞必须已经演变 在翻译失速期间保持阅读框的因素。随着核糖体似乎经常下降 压力，这些机制对于细胞存活和恢复至关重要。这个建议 专注于最近确定的机制，该机制涉及高度组成的多蛋白桥接 因子（MBF1）。我们的初步研究表明，该因素阻止了碰撞介导的+1帧。 这是MBF1结合的核糖体的初步cyoem结构，构成了我们的专业的基础 MBF1识别碰撞的核糖体以防止它们改变了阅读框的假设 领导一个。我们将通过三个目标检验这一假设。在第一个中，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MBF1的功能，以努力 建立核糖体碰撞与帧速率之间的关系。在第二个目标中，使用修改 核糖体促进方法评估整个转录组的帧速率，我们将剖析MBF1的作用 在防止在随机碰撞以及在压力下经历的帧中发生的帧。 第三个目的是使用一组电池进行MBF1募集到停滞核糖体的机制 生化和生物物理方法。我们最感兴趣地研究因素如何改变 功能和翻译机械的结构。总的来说，我们的跨学科方法建立和 扩展既定的专业知识和资源，我们计划用来揭示有关的重要细节 如何募集核糖体以调节其在压力下的功能。