Imaging Protein Synthesis on the Ribosome using Single-Molecule FRET

描述（由申请人提供）：翻译是蛋白质合成的过程，通过普遍保守的机制发生，该机制是所有生命领域基因表达的核心。翻译在人类细胞中受到高度调控，翻译控制的丧失是癌细胞生长的关键决定因素。细菌中的蛋白质合成是一系列临床重要抗生素的目标，这些抗生素用于对抗传染病。然而，对这些化合物的耐药性越来越普遍。核糖体是细胞翻译装置的主要组成部分，是调控的整合点。为了填补这一知识空白，核糖体功能的分子机制和翻译保真度将使用最先进的生物物理方法进行研究，包括单分子全反射荧光和零模波导成像技术。利用这些平台，将获得蛋白质合成过程中不同构象自由度的多维、高时空分辨率距离测量结果。在分子动力学模拟和低温电子显微镜领域的合作和互补努力下，这些研究将揭示翻译机制中结构事件的顺序和时间，以及它们如何促进定向和高保真蛋白质合成。长期目标是建立一个定量框架，将核糖体构象事件的微观速率常数与全局蛋白质合成联系起来。这将为翻译过程中决定速率的结构事件以及翻译保真度的分子基础提供新的线索，并将为理解核糖体调节的细胞机制和临床相关的翻译小分子效应的作用提供关键的见解。综合所获得的结果将提供关于精确聚焦的动态结构过程的新信息，支持翻译机制，并为探索在反应坐标中发生的特定事件如何靶向治疗提供平台。

DESCRIPTION (provided by applicant): Translation, the process of protein synthesis, occurs via a universally conserved mechanism that is central to gene expression in all domains of life. Translation is highly regulated in human cells and the loss of translation control is a key determinant of cancerous cell growth. Protein synthesis in bacteria is targeted by a broad array of clinically-important antibiotics that are used to combat infectious disease. However, resistance to these compounds is increasingly widespread. The ribosome is the principal component of the cellular translation apparatus and is the integration point for regulation. In order to fill this knowledge gap, the molecular mechanism of ribosome function and translational fidelity will be investigated using state-of-the-art biophysical methods, including single-molecule Total Internal Reflection Fluorescence and zero-mode waveguide imaging technologies. Using these platforms, the first multidimensional, high-spatial and -temporal resolution distance measurements of distinct conformational degrees of freedom will be obtained during both elemental and processive protein synthesis reactions. Together with collaborative and complementary efforts in the areas of molecular dynamics simulations and cryo-electron microscopy, these investigations will reveal the order and timing of structural events in translation machinery and how they contribute to driving directional and high-fidelity protein synthesis. The long-term goal is to establish a quantitative framework that relates the microscopic rate constants of conformational events in the ribosome to global protein synthesis. This will shed new light on the rate-determining structural events in the process as well as the molecular basis of translation fidelity, and will provide insights critical to understanding cellular mechanisms of ribosome regulation and the action of clinically-relevant small molecule effectors of translation. A synthesis of the results obtained will provide novel information about precisely focused, dynamic structural processes underpinning the translation mechanism and a platform for exploring how specific events that occur during the reaction coordinate may be targeted for therapeutic purpose.