Dynamics of Translation

项目概要（30行） 翻译是中心法则的终点，也是时空调控的起点， 基因表达。生物化学、生物物理学和结构学方法已经概述了 翻译的步骤，提供了一个菜单的关键因素，结构的核糖体和复合物， 以及引发、延伸和终止/再循环的基本步骤的动力学。 然而，关键步骤如起始、延伸和终止的机制， 它们是如何被RNA结构、修饰或调节蛋白调节的仍不清楚。 一个关键的挑战是翻译是高度动态的，涉及构象和组成 变化贯穿和遵循异质机制途径。在以往供资期间 我们将在这个MIRA合并的赠款支持期间，我们已经开发了单一的- 分子方法和试剂，观察翻译在真实的时间。我们将这些联合收割机 动态方法与cryoEM结构，以获得时间和详细的机械视图， 过程我们建议的研究重点是关键领域的翻译控制：如何启动是 在高等生物中实现-这里是一个小的（40 S）核糖体亚基被 与mRNA结合并识别启动子-将在酵母和人类中确定， 我们将探讨mRNA结构，蛋白质结合和修饰的核苷酸如何改变 过程我们将研究mRNA的5'端和3'端之间的长距离RNA相互作用是如何发生的。 可能对基础翻译起始及其控制至关重要。在延伸中，我们将继续 探索重新编码事件和共翻译蛋白质折叠，并开发观察方法 真核生物中的翻译延伸。我们将探讨核糖体的作用 停止/暂停并最终分流到核糖体质量控制途径中。最后我们将 了解正确的终止密码子被识别和核糖体再循环的途径 并确定正确与过早终止密码子在无意义中的区别 介导的衰变途径。我们的研究利用了数十年的试剂和方法 发展，和一个奇妙的合作者小组，探索翻译控制，及其 与人类健康和疾病的核心联系。

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.