Mutations in the highly conserved GGQ motif of class 1 polypeptide release factors abolish ability of human eRF1 to trigger peptidyl-tRNA hydrolysis

Abstract

Although the primary structures of class 1 polypeptide release factors (RF1 and RF2 in prokaryotes, eRF1 in eukaryotes) are known, the molecular basis by which they function in translational termination remains obscure. Because all class 1 RFs promote a stop-codon-dependent and ribosome-dependent hydrolysis of peptidyl-tRNAs, one may anticipate that this common function relies on a common structural motif(s). We have compared amino acid sequences of the available class 1 RFs and found a novel, common, unique, and strictly conserved GGQ motif that should be in a loop (coil) conformation as deduced by programs predicting protein secondary structure. Site-directed mutagenesis of the human eRF1 as a representative of class 1 RFs shows that substitution of both glycyl residues in this motif, G183 and G184, causes complete inactivation of the protein as a release factor toward all three stop codons, whereas two adjacent amino acid residues, G181 and R182, are functionally nonessential. Inactive human eRF1 mutants compete in release assays with wild-type eRF1 and strongly inhibit their release activity. Mutations of the glycyl residues in this motif do not affect another function, the ability of eRF1 together with the ribosome to induce GTPase activity of human eRF3, a class 2 RF. We assume that the novel highly conserved GGQ motif is implicated directly or indirectly in the activity of class 1 RFs in translation termination.

HemK, a class of protein methyl transferase with similarity to DNA methyl transferases, methylates polypeptide chain release factors, and hemK knockout induces defects in translational termination

“…Because the class 1 RFs are essential and their structure is highly conserved in all eubacteria and genome-containing organelles (23,35), it is likely that conserved HemK homologs play the same role as in E. coli. Consistent with this idea, at least one HemK homolog is encoded in all of the eubacterial and eukaryotic genomes that have been completely sequenced.…”

The methylator meets the terminator

“…In E. coli, there are two RFs whose structures mimic tRNAs as they recognize the UGA, UAA, and UAG terminator codons in the A site of the ribosome and allow the hydrolysis of the nascent peptide chain from the last tRNA at the peptidyl transferase center (7). The most highly conserved feature of RFs from eukaryotic and prokaryotic organisms is a GGQ sequence, located near the site of the peptidyl-tRNA ester bond that is cleaved in the termination reaction (7,8). In 2000, Dincbas-Renqvist et al (9) did a careful chemical, chromatographic, and mass spectral analysis of RF2 from E. coli and provided evidence that the conserved glutamine residue at position 252 in this sequence is posttranslationally modified by a methylation reaction to give the N-5-methylglutamine derivative ( Fig.…”

Accommodating the bacterial decoding release factor as an alien protein among the RNAs at the active site of the ribosome

“…While it was tempting to speculate that this was a rare non-physiological form (a crystallization artefact), the same structure was resolved from crystals with different unit cells, and later the second bacterial factor, RF-1, was shown to have the same overall structure. Puzzlingly, two motifs characterized biochemically and genetically as likely to be involved at the DC [28] and the enzyme centre, respectively [29], were quite close together (27 Å) in the structure and could not span the two The release factor at the ribosomal active centre 594 npg parts of the ribosomal active centre. One of the motifs, the tripeptide sequence GGQ [29] (the only sequence in common between the prokaryotic and eukaryotic factors), was expected to be at the PTC, whereas the other motif, PXT in RF1 and SPF in RF2 [28] (proposed as the 'anticodon' responsible for discrimination of the second and third bases of the stop codon after an elegant series of genetic studies that achieved codon switching dependent on these bases), was expected to be at the DC.…”