Peptidyl transferase center activity observed in single ribosomes

We demonstrate the functional activity of single ribosomal complexes, opening the way for detailed studies of the trajectories of protein synthesis. Our approach employs a single-molecule detection system, capable of picoseconds to minutes resolution, to observe a growing peptide labeled at its N terminus with the fluorophore tetramethylrhodamine (TMR). Single complexes of mRNA-programmed ribosomes with TMR-Met-tRNA f Met or TMR-Met-Phe-tRNA Phe are immobilized on mica and observed by fluorescence. Immobilized ribosome·mRNA·TMR-Met-tRNA f Met complexes form peptide bonds with puromycin. Single-molecule detection reveals dynamics on the scale of seconds at the ribosomal peptidyl transferase center.