A Conserved Base-pair between tRNA and 23 S rRNA in the Peptidyl Transferase Center Is Important for Peptide Release

The 3′ terminus of tRNAs has the universally conserved bases C74C75A76 that interact with the ribosomal large subunit. In the ribosomal P site, bases C74 and C75 of tRNA, form Watson–Crick base-pairs with G2252 and G2251, respectively, present in the conserved P-loop of 23 S rRNA. Previous studies have suggested that the G2252-C74 base-pair is important for peptide bond formation. Using a pure population of mutant ribosomes, we analyzed the precise role of this base-pair in peptide bond formation, elongation factor G-dependent translocation, and peptide release by release factor 1. Surprisingly, our results show that the G2252-C74 base-pair is not essential for peptide bond formation with intact aminoacyl tRNAs as substrates and for EF-G catalyzed translocation. Interestingly, however, peptide release was reduced substantially when base-pair formation between G2252 and C74 of P site tRNA was disrupted, indicating that this conserved base-pair plays an important role in ester bond hydrolysis during translation termination.