Imaging Protein Synthesis on the Ribosome using Single-Molecule FRET

描述(申请人提供)：翻译，蛋白质合成的过程，通过一种普遍保守的机制发生，这是生命所有领域中基因表达的中心。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靶点，这些抗生素被用来对抗传染病。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，也是调节的整合点。为了填补这一知识空白，将利用最先进的生物物理方法，包括单分子全内反射荧光和零模波导成像技术，研究核糖体功能和翻译保真度的分子机制。利用这些平台，将首次在基本和过程蛋白质合成反应中获得不同构象自由度的多维、高空间和时间分辨率的距离测量。再加上分子动力学模拟和冷冻电子显微镜领域的合作和互补努力，这些研究将揭示翻译机器中结构事件的顺序和时间，以及它们如何有助于推动定向和高保真蛋白质合成。长期目标是建立一个量化框架，将核糖体中构象事件的微观速率常数与全球蛋白质合成联系起来。这将揭示这一过程中决定速率的结构事件以及翻译保真度的分子基础，并将为理解核糖体调节的细胞机制和与临床相关的翻译小分子效应器的作用提供关键的见解。对所获得结果的综合将提供关于支持翻译机制的精确聚焦的动态结构过程的新信息，并为探索在反应协调期间发生的特定事件如何针对治疗目的提供一个平台。

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.