CAREER: Exploring Conservation in the Molecular Determinants of the Fidelity Mechanism in Translation

本综合研究和教育计划的重点是单分子荧光共振能量转移FRET（smFRET）方法的应用和发展，以研究核糖体催化蛋白质合成动态过程的作用和功能。重点将是描述不同物种核糖体上tRNA选择和易位过程中核糖体内纳米尺度重塑事件的结构和动力学参数，以及这些随机事件如何促进保真度的机制。我们的主要目的是确定这些机制在进化过程中的保守和分歧特征。smFRET成像方法的应用，结合遗传、生化和计算的努力，将提供高空间和时间分辨率的核糖体能量景观的定量测量。为了实现这一目标，将在生物相容性微流体系统的构建、单分子数据分析所需的计算工具的设计以及荧光染料分子的稳定方面取得进展。该项目的成功整合和完成有望促进对蛋白质合成的保守机制的理解。对核糖体内动态结构过程作用的深入了解将产生广泛的影响，跨越任何构象变化影响细胞功能和调节的酶学系统。这项研究的性质为年轻科学家提供了一个独特的培训环境，需要新的技术和跨学科的专业知识来有效地从事他们的科学追求。

This integrated research and educational plan focuses on the application and development of single-molecule Fluorescence Resonance Energy Transfer FRET(smFRET) methods to investigate the role and function of dynamic process of protein synthesis catalyzed by the ribosome. Particular focus will be given to delineating the structural and kinetic parameters of nanometer-scale remodeling events within the ribosome during tRNA selection and translocation processes on ribosomes from different species, and how these stochastic events contribute to the mechanism of fidelity. Our principal aim is to determine conserved and divergent features of these mechanisms across evolution. The application of smFRET imaging methods, in combination with genetic, biochemical, and computation efforts, will provide quantitative measures of the ribosome energy landscape at high-spatial and -time resolution. Towards this goal, advances will be made in the construction of biologically compatible microfluidic systems, the design of computational tools required for the analysis of single-molecule data, and in the stabilization of fluorescent dye molecules. The successful integration and completion of this project is expected to advance the understanding of conserved mechanisms in protein synthesis across evolution. A deeper knowledge of the role of dynamic structural processes within the ribosome will have a broad impact, spanning any enzymological system where conformational changes impact function and regulation in the cell. The nature of this research provides a unique training environment for young scientists where new technologies and cross-disciplinary expertise is required to engage effectively their scientific pursuits.