Protein synthesis on single ribosomes

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The research will test crucial functional aspects of mRNA-directed ribosomal protein biosynthesis at the level of individual ribosomes. Two new methodological developments, single molecule fluorescence polarization and force-feedback infrared laser optical trap will be applied, providing information currently unavailable about the mechanism of the ribosomal elongation cycle. Fluorescent probes will be inserted into elongation factors (EFs) and transfer RNA (tRNA) with known, predetermined orientations. The mechanism of proof-reading the genetic code and of translocation along the mRNA will be determined by investigating the kinetics, and angular relationships between, structural changes in EFs and tRNA. Whether EFs are motors or switches will be elucidated. The timing of tRNA motions through the ribosome will be compared to those of structural changes in the EFs. Using the optical trap, the relationship between mechanical tension in mRNA and velocity of peptide elongation (the force-velocity curve) of single ribosomes will be determined. The influence of altering substrate and product concentrations on the force velocity curve will be used in distinguishing hypothetical mechanisms of translocation. Protein synthesis is ubiquitous among living organism and the similarities between all ribosomes indicate that the elongation cycle is one of the most fundamental biological processes. Thus the collaboration proposed in this application between laboratories having expertise in motor protein and ribosome function, in vitro single molecule mechanics, and novel polarization spectroscopy will advance the understanding of ribosomal and G-protein function, and should impact very broadly in biophysics and biomedicine.