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.
This NSF CAREER award enabled the first quantitative single-molecule Fluorescence Resonance Energy Transfer FRET (smFRET) investigations comparing conserved and divergent biophysical features of the mechanisms employed by bacterial and eukaryotic ribosomes -the macromolecular complexes responsible for catalyzing protein synthesis in the cell. This award contributed to the generation of more than 8 peer-reviewed research and review articles, encyclopedia and book chapters, approximately 30 public lectures at major conferences and academic research institutions across the United States and abroad as well as the advancement of the scientific training of high-school, undergraduate and graduate students. This award also enabled educational outreach efforts that increased awareness in elementary students in the greater New York City area about what it means to be a scientist and that "Science is All Around Us". The broader impact of the research conducted includes a deeper understanding of the fundamental role that spontaneous (thermally accessible) dynamic processes play in the function and regulation of the protein synthesis machinery across all domains of life. The findings obtained also shed important new light on distinct mechanistic features of the translation machinery in prokaryotic and eukaryotic cells as well as insights into how environmental cues -including small-molecule metabolites - can contribute to the cellular regulation of protein synthesis in a species-specific manner.
