Ribosome-catalyzed peptide-bond formation with an A-site substrate covalently linked to 23S ribosomal RNA

In the ribosome, the aminoacyl-transfer RNA (tRNA) analog 4-thio-dT-p-C-p-puromycin crosslinks photochemically with G2553 of 23S ribosomal RNA (rRNA). This covalently linked substrate reacts with a peptidyl-tRNA analog to form a peptide bond in a peptidyl transferase-catalyzed reaction. This result places the conserved 2555 loop of 23S rRNA at the peptidyl transferase A site and suggests that peptide bond formation can occur uncoupled from movement of the A-site tRNA. Crosslink formation depends on occupancy of the P site by a tRNA carrying an intact CCA acceptor end, indicating that peptidyl-tRNA, directly or indirectly, helps to create the peptidyl transferase A site.