Analysis of mutations at residues A2451 and G2447 of 23S rRNA in the peptidyltransferase active site of the 50S ribosomal subunit

On the basis of the recent atomic-resolution x-ray structure of the 50S rib osomal subunit, residues A2451 and G2447 of 235 rRNA were proposed to parti cipate directly in ribosome-catalyzed peptide band formation. We have exami ned the peptidyltransferase and protein synthesis activities of ribosomes c arrying mutations at these nucleotides. In Escherichia toll, pure mutant ri bosome populations carrying either the G2447A or G2447C mutations maintaine d cell viability. In vitro, the G2447A ribosomes supported protein synthesi s at a rate comparable to that of wild-type ribosomes. In single-turnover p eptidyltransferase assays, G2447A ribosomes were shown to have essentially unimpaired peptidyltransferase activity at saturating substrate concentrati ons. All three base changes at the universally conserved A2451 conferred a dominant lethal phenotype when expressed in E, toll. Nonetheless, significa nt amounts of 2451 mutant ribosomes accumulated in polysomes, and all three 2451 mutations stimulated frameshifting and readthrough of stop codons in vivo. Furthermore, ribosomes carrying the A2451U transversion synthesized f ull-length beta -lactamase chains in vitro. Pure mutant ribosome population s with changes at A2451 were generated by reconstituting Bacillus stearothe rmophilus 50S subunits from in vitro transcribed 235 rRNA. In single-turnov er peptidyltransferase assays, the rate of peptide bond formation was dimin ished 3- to 14-fold by these mutations. Peptidyltransferase activity and in vitro beta -lactamase synthesis by ribosomes with mutations at A2451 or G2 447 were highly resistant to chloramphenicol. The significant levels of pep tidyltransferase activity of ribosomes with mutations at A2451 and G2447 ne ed to be reconciled with the roles proposed for these residues in catalysis .