The principal component of the translation machinery, and the integration point for regulation of protein synthesis is the ribosome, a two-subunit, RNA-protein enzyme. In concert with RNA and protein translation factors, the ribosome converts messenger RNA (mRNA) into a specific protein sequence. Translation is highly regulated in normal cell physiology;loss of translation control is a key determinant of cell growth in the cancerous state. Translation is also targeted by a broad array of clinically useful therapeutic compounds important for the treatment of infectious diseases. Recent breakthroughs in structural biology have yielded spectacular snapshot images of the ribosome and its components. These data provide the foundation for a much needed physical framework upon which genetic, biochemical and biophysical investigations of ribosome function must be reconciled. To this end, we propose to study the mechanism of tRNA selection and translocation processes on the single-molecule scale. The hypothesis central to the proposed research is that translation fidelity stems from conformational changes within the ribosome that are triggered by its interaction with tRNA and translation factors. Using state-of-the-art Total Internal Reflection Fluorescence techniques we will extract the global and microscopic rate constants of the dynamic structural changes of the translation machinery from high-spatial and -time resolution distance measurements. These data, unobtainable in ensemble studies, will aid in the elucidation of the basic translation mechanism. They will serve as a platform for the investigation of translational regulation mechanisms within the cell, and for screening novel compounds which affect specific aspects of ribosome function. To test our hypothesis, we will probe the tRNA selection and translocation mechanisms using single-molecule FRET (smFRET) as a tool to make direct measurements of the dynamic, structural processes within the functioning ribosome in a reconstituted translation extract. Specifically, we propose:
AIM 1 ] Measure the order and timing of tRNA motions on the ribosome during Elongation Factor-Tu (EF-Tu)-mediated tRNA selection and Elongation Factor-G (EF-G)-dependent translocation.
AIM 2 ] Determine how movements and conformational changes of EF- Tu, and EF-G relate to the mechanisms of aa-tRNA selection and translocation, respectively.
AIM 3 ] Delineate the influence of conformational changes of the ribosome during protein synthesis on the motions of tRNA during tRNA selection and translocation. The instruments and methodologies developed through this research may also be applicable to investigations of other molecular assemblies where compositional and conformational processes play a role in biological function.
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