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

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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.