Crystal Structure of the 70S Ribosome Bound with the Q253P Mutant Form of Release Factor RF2

Abstract

Bacterial translation termination is mediated by release factors RF1 and RF2, which recognize stop codons and catalyze hydrolysis of the peptidyl-tRNA ester bond. The catalytic mechanism has been debated. We proposed that the backbone amide NH group, rather than the side chain, of the glutamine of the universally conserved GGQ motif participates in catalysis by H-bonding to the tetrahedral transition-state intermediate and by product stabilization. This was supported by complete loss of RF1 catalytic activity when glutamine is replaced by proline, the only residue that lacks a backbone NH group. Here, we present the 3.4 Å crystal structure of the ribosome complex containing the RF2 Q253P mutant and find that its fold, including the GGP sequence, is virtually identical to that of wild-type RF2. This rules out proline-induced misfolding, and further supports the proposal that catalytic activity requires interaction of the Gln253 backbone amide with the 3′ end of peptidyl-tRNA.

Structural Basis for Translation Termination on a Pseudouridylated Stop Codon

“…1d, 1e). The ribosomal 50S and 30S subunits adopt the non-rotated (classical) conformation, similar to that observed in structures of termination complexes formed with the canonical (non-modified) stop codons [9–12]. The conformation of the release factor (Fig.…”

“…Because the peptidyl-transferase centers of archaeal and eukaryotic ribosomes do not contain an L27 homolog and the mechanism of peptidyl transfer is universally conserved, bacterial L27 is unlikely to be critical for peptidyl transfer. L27 might, however, contribute to bacterial translation termination, which is mechanistically distinct from that in eukaryotes, as indicated by sequence and structure divergence between bacterial and eukaryotic release factors RF1/2 and eRF1, different mechanisms of stop codon recognition [9–12, 15, 16], and requirement for a translational GTPase eRF3 in eukaryotes [28–32]. …”

Uniformity of Peptide Release Is Maintained by Methylation of Release Factors

“…In all previous structures of Tth RFs bound to the 70S, domain three closely packs against 23S rRNA nucleotides of the PTC (Figure 4A) (Jin et al, 2010; Korostelev et al, 2008a; Santos et al, 2013; Weixlbaumer et al, 2008). The universally conserved GGQ motif is located at the tip of domain three and the glutamine (Q235 in RF1, Q252 in RF2) backbone amide is located in an equivalent position at the 2’ - end of the A-site tRNA as previously reported (Figure 4B, Figure S1).…”

“…The universally conserved GGQ motif is located at the tip of domain three and the glutamine (Q235 in RF1, Q252 in RF2) backbone amide is located in an equivalent position at the 2’ - end of the A-site tRNA as previously reported (Figure 4B, Figure S1). (Jin et al, 2010; Korostelev et al, 2008a; Santos et al, 2013; Weixlbaumer et al, 2008). The structures of the 70S-RF1 Me with and without the Q235 methylation reveal that the overall location of the backbone of the GGQ motif is unaffected by the modification.…”

Peptide release promoted by methylated RF2 and ArfA in nonstop translation is achieved by an induced-fit mechanism

“…In all the canonical termination complexes, the main chain amide group is within hydrogen bond distance to the catalytic water, suggesting that it directly coordinates the water for the hydrolytic reaction in releasing the nascent peptide from the P-tRNA. Furthermore, it has been demonstrated that a substitution of a glutamine by a proline abolished the peptide release activity by RF2 without deforming the overall conformation of the GGQ motif in the PTC of the ribosome (Santos et al 2013). In addition to the main chain amide group, the side chain of the glutamine has been implicated in providing important packing for specifically selecting a water molecule as the nucleophile for the release reaction, as proposed from a seminal biochemical investigation (Shaw and Green 2007).…”