Mutations in the conserved P loop perturb the conformation of two structural elements in the peptidyl transferase center of 23 S ribosomal RNA

Evidence is presented for the participation of the P loop (nucleotides G225 0-C2254) of 23 S rRNA in establishing the tertiary structure of the peptidy l transferase center. Single base substitutions were introduced into the P loop, which participates in peptide bond formation through direct interacti on with the CCA end of P site-bound tRNA. These mutations altered the patte rn of reactivity of RNA to chemical probes in a structural subdomain encomp assing the P loop and extending roughly from G2238 to A2433. Most of the ef fects on chemical modification in the P loop subdomain occurred near sites of tertiary interactions inferred from comparative sequence analysis, indic ating that these mutations perturb the tertiary structure of this region of RNA. Changes in chemical modification were also seen in a subdomain compos ed of the 2530 loop (nucleotides G2529-A2534) and the A loop (nucleotides U 2552-C2556), the latter a site of interaction with the CCA end of A site-bo und tRNA. Mutations in the P loop induced effects on chemical modification that were commensurate with the severity of their characterized functional defects in peptide bond formation, tRNA binding and translational fidelity. These results indicate that, in addition to its direct role in peptide bon d formation, the P loop contributes to the tertiary structure of the peptid yl transferase center and influences the conformation of both the acceptor and peptidyl tRNA binding sites. (C) 1999 Academic Press.