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

Abstract

On the basis of the recent atomic-resolution x-ray structure of the 50S ribosomal subunit, residues A2451 and G2447 of 23S rRNA were proposed to participate directly in ribosome-catalyzed peptide bond formation. We have examined the peptidyltransferase and protein synthesis activities of ribosomes carrying mutations at these nucleotides. In Escherichia coli, pure mutant ribosome populations carrying either the G2447A or G2447C mutations maintained cell viability. In vitro, the G2447A ribosomes supported protein synthesis at a rate comparable to that of wild-type ribosomes. In single-turnover peptidyltransferase assays, G2447A ribosomes were shown to have essentially unimpaired peptidyltransferase activity at saturating substrate concentrations. All three base changes at the universally conserved A2451 conferred a dominant lethal phenotype when expressed in E. coli. Nonetheless, significant 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 full-length ␤-lactamase chains in vitro. Pure mutant ribosome populations with changes at A2451 were generated by reconstituting Bacillus stearothermophilus 50S subunits from in vitro transcribed 23S rRNA. In single-turnover peptidyltransferase assays, the rate of peptide bond formation was diminished 3-to 14-fold by these mutations. Peptidyltransferase activity and in vitro ␤-lactamase synthesis by ribosomes with mutations at A2451 or G2447 were highly resistant to chloramphenicol. The significant levels of peptidyltransferase activity of ribosomes with mutations at A2451 and G2447 need to be reconciled with the roles proposed for these residues in catalysis.

Comprehensive genetic selection revealed essential bases in the peptidyl-transferase center

“…4B). SSER showed that, besides the wild-type triad (A2451-G2447-G2061), only A2451-A2447-G2061 was selected as a functional variant, as a slow-growth phenotype (Table 1), consistent with previous observations that G in 2447 can be replaceable to A (25). The only deviation from our finding is the viable A2451T found in Mycobacterium smegmatis (26).…”

“…This discrepancy might be explained by the use a high-copy plasmid with ColE1 origin to rescue the E. coli ⌬rrn7 strain (25), whereas we used a low-copy (Ϸ5 copies) plasmid with a pSC101 origin (41), thus requiring a high level of ribosomal functionality. Supporting this view is the apparent reduction of viability (relative growth rate of only 62%, Table 1) shown by our G2447A variant, in contrast to the G2447A mutant that has a similar growth phenotype to that of wild-type cells (25). This observation may, therefore, indicate that G2447 plays a role in maintaining the critical H bond network in the PTC.…”

“…The complex H bond network between essential bases consisting of G2061, C2063, A2450, A2451, C2452, C2501, and U2504 may provide the molecular stage for protein synthesis. It has been reported that G2447 can be replaced with A or C in E. coli ⌬rrn7 strain (25). G2447 forms a connection with A2451 and A2450.…”

“…Consistently, the hypothesis, based on structures of H50S complexed with minimum substrates, that the PTC acts as a general acid-base catalyst (2,(20)(21)(22), was contradicted by various mutagenesis and biochemical studies (12,(23)(24)(25)(26)(27)(28). The main catalytic contribution of the ribosomes, substrates positioning at proper orientation (4,28,29), is achieved by remote interactions, accompanied by symmetrical base-pairing of C75 of both tRNAs with G2553 (Escherichia coli numbering throughout) and G2251 (Fig.…”

The transition state for peptide bond formation reveals the ribosome as a water trap

“…Recent years have witnessed considerable progress in our understanding of the peptidyl transfer process due to high-resolution crystallographic structures of the large ribosomal subunit with transition state (TS) analogs (1), as well as kinetic measurements (2)(3)(4)(5)(6)(7)(8)(9), mutagenesis data (4,(10)(11)(12)(13)(14), and computational studies (15)(16)(17). Hence, the current model of the peptidyl transfer reaction is that ribosomal nucleobases are not directly involved in bond making or breaking through acid-base catalysis (4,10,11,13,18,19), but that the A76 2′-OH group of the P-site substrate plays a key role in mediating proton transfer from the attacking nucleophile to the leaving 3′ ester oxygen (1,15,(20)(21)(22). Furthermore, it was shown that the lower free energy barrier for the ribosome reaction, compared to an uncatalyzed reference reaction in water, is entirely due to a less negative activation entropy (3,6,7).…”