The main focus of our proposal is the time-resolved investigation of molecular mechanisms of protein biosynthesis. We have demonstrated the functional activity of single ribosomal complexes via a novel single molecule spectroscopic technique, namely confocal optical microscopy. Using confocal microscopy we are able, on a picoseconds to minutes timescale, to observe a growing peptide labeled at its N-terminus with the fluorophore tetramethyl-rhodamine (TMR). Single complexes of mRNA-programmed ribosomes with TMR-Met-tRNAfMet or TMR-Met-Phe-tRNAPhe are immobilized on a mica plate and observed by fluorescence. Immobilized ribosomemRNATMR-Met-tRNAfMet complexes form peptide bonds with puromycin. Single-molecule detection has revealed dynamics on the scale of seconds at the ribosomal peptidyl transferase center.
NIH ExPorter
NIH ExPorter