Structure of the 70S ribosome bound to release factor 2 and a substrate analog provides insights into catalysis of peptide release

Abstract

We report the crystal structure of release factor 2 bound to ribosome with an aminoacyl tRNA substrate analog at the ribosomal P site, at 3.1 Å resolution. The structure shows that upon stopcodon recognition, the universally conserved GGQ motif packs tightly into the peptidyl transferase center. Nucleotide A2602 of 23S rRNA, implicated in peptide release, packs with the GGQ motif in release factor 2. The ribose of A76 of the peptidyl-tRNA adopts the C2′-endo conformation, and the 2′ hydroxyl of A76 is within hydrogen-bond distance of the 2′ hydroxyl of A2451. The structure suggests how a catalytic water can be coordinated in the peptidyl transferase center and, together with previous biochemical and computational data, suggests a model for how the ester bond between the peptidyl tRNA and the nascent peptide is hydrolyzed.ribosome structure | translational termination | X-ray crystallography T he ribosome translates the genetic information present in mRNA into proteins (1). A crucial step of this process is the termination of protein synthesis, which involves the cleavage and release of the nascent peptide chain from the P-site tRNA when the end of the coding sequence is reached. Translational termination by the ribosome is a precise and complex step that occurs when a stop codon of an mRNA is encountered in the A site of the small ribosomal subunit. The stop codons are recognized by proteins called class I release factors (RFs). In bacteria, two class I RFs recognize the three stop codons with overlapping specificity: RF1 recognizes UAG and RF2 recognizes UGA, whereas both factors recognize UAA (2). Upon stop-codon recognition, the class I RF promotes the hydrolysis of the ester bond between the nascent polypeptide and the peptidyl tRNA at the ribosomal P site, leading to the release of the nascent polypeptide and the termination of protein synthesis (3). Eukaryotes and archaea possess a single "omnipotent" class I RF, eRF1 or aRF1, respectively, which recognizes all three stop codons (4, 5). eRF1 and aRF1 are highly homologous with each other and were proposed to have evolved independently from their bacterial counterpart (4).A major advance in our understanding in the specificity of stop-codon recognition and peptide release at the molecular level was achieved when three high-resolution crystal structures of the 70S ribosome complexed with class I RFs and their cognate stop codons were solved at atomic resolution (6-8). An analysis of the interactions of RF1 and RF2 with the decoding center in these structures provides explanations for the specificity of stop-codon recognition by these factors. However, because all three structures represent the product state with a deacylated tRNA in the P site, the mechanism of peptide release is less clear.A universally conserved GGQ motif (9) that was shown to be required for catalytic activity (10, 11) was indeed found in the peptidyl transferase center (PTC) in earlier structures by cryoEM (12, 13) or crystallography at ∼6 Å (14). Mutations introduced at the first two co...

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

“…The same modification is also found in Saccharomyces cerevisiae and is required for optimal cell growth (Heurgué-Hamard et al 2005). The packing role of the side chain of the glutamine for the catalysis of peptide release has been demonstrated biochemically (Shaw and Green 2007), structurally (Jin et al 2010), and computationally Aqvist 2007, 2009). The enhanced packing of the N 5 -methylation of the catalytic glutamine in the GGQ motif has been suggested by computational studies (Trobro and Aqvist 2007).…”

“…The interactions of RF2 with its cognate stop codons in the decoding center revealed by the crystal structures offers molecular explanations for the specificity of stop codon recognition in the ribosome Weixlbaumer et al 2008). For the peptide release, the catalytic GGQ motif in the release factors makes extensive interactions in the PTC Weixlbaumer et al 2008;Jin et al 2010). This catalytic motif is positioned in the PTC by a network of hydrogen bond interactions between highly conserved residues in release factors and nucleotides in the 23S rRNA (Jin et al 2010).…”

“…Crystal structures of the 70S ribosome complexed with class I release factors and their cognate stop codons, which have been solved at a near-atomic resolution Laurberg et al 2008;Weixlbaumer et al 2008;Jin et al 2010), have provided us a clear picture of the translational termination in three dimensions. The interactions of RF2 with its cognate stop codons in the decoding center revealed by the crystal structures offers molecular explanations for the specificity of stop codon recognition in the ribosome Weixlbaumer et al 2008).…”

“…For the peptide release, the catalytic GGQ motif in the release factors makes extensive interactions in the PTC Weixlbaumer et al 2008;Jin et al 2010). This catalytic motif is positioned in the PTC by a network of hydrogen bond interactions between highly conserved residues in release factors and nucleotides in the 23S rRNA (Jin et al 2010). The conformation of the glycines in the GGQ motif facilitates proper placement of the conserved glutamine in the core of the PTC, where it forms a densely packed pocket with the A76 of the peptidyl tRNA, and A2451, C2063, U2585, and U2506 of the 23S rRNA for the catalysis of the peptide release.…”

Another burst of smoke: Atomic resolution structures of RF3 bound to the ribosome

“…Crystal structures with RF1 or RF2 bound to pre-or post-termination ribosomes are unambiguously in a classical, nonratcheted state Laurberg et al 2008;Weixlbaumer et al 2008;Jin et al 2010). As such, RF3 may initially bind the ribosome in the classical state, or perhaps post-termination ribosome complexes sample a ratcheted state.…”