Dynamics of Translation

项目总结(30行) 翻译是翻译的中心教条的终点和时间和空间的调整点 基因表达。生化、生物物理和结构方法概述了一般的 翻译步骤，提供关键因素的菜单，核糖体和复合体的结构， 以及引发、延伸和终止/再循环这几个基本步骤的动力学。 尽管如此，启动、延伸和终止等关键步骤的机制，以及 它们是如何由RNA结构、修饰或调节蛋白调控的，目前尚不清楚。 一个关键的挑战是翻译是高度动态的，涉及构象和构词 在不同的机械路径中和之后的变化。在之前的资助期间 在我们将在这一米拉合并的赠款的支持下，我们开发了单一- 实时观察翻译的分子方法和试剂。我们把这些结合在一起 使用低温EM结构的动力学方法，以获得时间和详细的机械视图 这一过程。我们建议的研究集中在翻译控制的关键领域：启动是如何 在高等生物体中实现的-这里是小的(40s)核糖体亚基被 结合到一个信使核糖核酸和识别一个起点-将在酵母和人类中确定，和 我们将探索信使核糖核酸结构、蛋白质结合和修饰核苷酸如何改变 进程。我们将研究mRNAs 5‘和3’端之间的长程RNA相互作用 可能是基础翻译启动及其控制的关键。在延伸方面，我们将继续 探索记录事件和共翻译蛋白质折叠，并开发观看方法 真核生物中的翻译延伸。我们将探索核糖体的作用 停顿/停顿并最终分流到核糖体质量控制途径。最后，我们会 了解正确的终止密码子被识别和核糖体回收的途径 并确定在无稽之谈中如何区分正确的和过早的终止密码子 介导的衰变途径。我们的研究利用了数十年的试剂和方法 开发，以及一组出色的合作者来探索翻译控制，以及它 与人类健康和疾病的中心联系。

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.