Peptidyl transferase center activity observed in single ribosomes

We demonstrate the functional activity of single ribosomal complexes, openi ng the way for detailed studies of the trajectories of protein synthesis. O ur approach employs a single-molecule detection system, capable of picoseco nds to minutes resolution, to observe a growing peptide labeled at its N te rminus with the fluorophore tetramethylrhodamine (TMR). Single complexes of mRNA-programmed ribosomes with TMR-Met-tRNA(f)(Met) or TMR-Met-Phe-tRNA(Ph e) are immobilized on mica and observed by fluorescence. Immobilized riboso me . mRNA . TMR-Met-tRNA(f)(Met) complexes form peptide bonds with puromyci n. Single-molecule detection reveals dynamics on the scale of seconds at th e ribosomal peptidyl transferase center. (C) 1999 Academic Press.