Dynamics of Translation

翻译动力

• 批准号: 10617792
• 负责人: JOSEPH D PUGLISI
• 金额: $ 76.7万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-06-01 至 2027-05-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10617792
• 关键词: Area; Binding; Binding Proteins; Biochemical; Biochemistry; Biophysics; Code; Complex; Cryoelectron Microscopy; Disease; Event; Funding; Gene Expression; Genetic; Grant; Health; Human; Kinetics; Messenger RNA; Methods; Modification; Molecular Conformation; Nonsense Codon; Nonsense-Mediated Decay; Nucleotides; Organism; Pathway interactions; Process; Quality Control; RNA; Reagent; Recycling; Regulation; Research; Ribosomes; Role; Structure; Terminator Codon; Time; Translation Initiation; Translations; Yeasts; genetic regulatory protein; method development; protein folding; single molecule; structural biology

PROJECT SUMMARY (30 lines) Translation is the endpoint of the central dogma and point of temporal and spatial regulation in gene expression. Biochemical, biophysical and structural methods have outlined the general steps of translation, providing a menu of key factors, structures of ribosomes and complexes, and kinetics for the essential steps of initiation, elongation and termination/recycling. Nonetheless, the mechanisms of key steps such as initiation, elongation and termination, and how they are regulated by RNA structures, modification or regulatory proteins remains unclear. A key challenge is that translation is highly dynamic, involving conformational and compositional changes throughout and following heterogeneous mechanistic pathways. During prior funding periods supported by the grants that we will merge in this MIRA, we have developed single- molecule approaches and reagents that observe translation in real time. We combine these dynamic methods with cryoEM structures to gain a temporal and detailed mechanistic view of the process. Our proposed research focuses on key areas translational control: how initiation is achieved in higher organisms—here the pathway by which a small (40S) ribosomal subunit is bound to a mRNA and recognizes a start—will be determined in both yeast and humans, and we will explore how mRNA structure, protein binding and modified nucleotides change the process. We will investigate how long-range RNA interaction between 5’ and 3’ ends of mRNAs may be critical for basal translation initiation and its control. In elongation, we will continue to explore recoding events and co-translational protein folding and develop methods to watch translation elongation in eukaryotic organisms. We will explore the role of ribosomal stalling/pausing and eventual shunting into ribosomal quality control pathways. Finally, we will understand the pathways by which correct stop codons are recognized and ribosomes recycled and determine how correct vs premature stop codons are distinguished in the nonsense mediated decay pathway. Our research leverages decades of reagent and methods development, and a wonderful group of collaborators to explore translational control, and its central linkage to human health and disease.

项目摘要（30 行） 翻译是中心法则的终点和时空调节点 生物化学、生物物理和结构方法概述了基因表达的一般方法。 翻译步骤，提供关键因素、核糖体和复合物结构的菜单， 以及引发、延伸和终止/回收的基本步骤的动力学。 然而，诸如起始、延伸和终止等关键步骤的机制以及 它们如何受到 RNA 结构、修饰或调节蛋白的调节仍不清楚。 一个关键的挑战是翻译是高度动态的，涉及构象和组合 在之前的资助期间，贯穿整个变化并遵循异质的机制路径。 在我们将合并到这个 MIRA 中的赠款支持的时期，我们开发了单- 我们将实时观察翻译的分子方法和试剂结合起来。 使用冷冻电镜结构的动态方法可以获得时间和详细的机械视图 我们提出的研究重点是翻译控制的关键领域：启动是如何进行的。 在高等生物体中实现——这里是一个小（40S）核糖体亚基的途径 与 mRNA 结合并识别起始点——将在酵母和人类中确定，并且 我们将探索 mRNA 结构、蛋白质结合和修饰核苷酸如何改变 我们将研究 mRNA 5' 和 3' 末端之间的长程 RNA 相互作用。 可能对于基础翻译起始及其控制至关重要，我们将继续研究。 探索记录事件和共翻译蛋白质折叠并开发观察方法 我们将探讨核糖体的翻译延伸。 最后，我们将停止/暂停并最终分流到核糖体质量控制途径。 了解正确终止密码子被识别和核糖体回收的途径 并确定如何区分无意义的正确终止密码子和过早终止密码子 我们的研究利用了数十年的试剂和方法。 开发，以及一群出色的合作者来探索翻译控制及其 与人类健康和疾病的中心联系。