Single amino acid substitution in prokaryote polypeptide release factor 2 permits it to terminate translation at all three stop codons (translation terminationystop codon recognitionydiscriminatorytRNA mimicry)

Prokaryotic translational release factors, RF1 and RF2, catalyze polypeptide release at UAGyUAA and UGAyUAA stop codons, respectively. In this study, we isolated a bacterial RF2 mutant (RF2*) containing an E167K substi- tution that restored the growth of a temperature-sensitive RF1 strain of Escherichia coli and the viability of a chromosomal RF1yRF2 double knockout. In both in vivo and in vitro polypeptide termination assays, RF2* catalyzed UAGyUAA termination, as does RF1, as well as UGA termination, show- ing that RF2* acquired omnipotent release activity. This result suggests that the E167K mutation abolished the putative third-base discriminator function of RF2. These findings are interpreted as indicating that prokaryotic and eukaryotic release factors share the same anticodon moiety and that only one omnipotent release factor is sufficient for bacterial growth, similar to the eukaryotic single omnipotent factor. The termination of protein synthesis takes place on the ribosomes as a response to a stop, rather than a sense, codon in the ''decoding'' site (A site). Translation termination gen- erally requires two codon-specific polypeptide release factors (RFs), RF1 (for UAGyUAA) and RF2 (for UGAyUAA), in prokaryotes (1, 2) and one factor, eRF1 (omnipotent for the three stop codons), in eukaryotes (2-4) (Fig. 1A). However, the mechanism of stop codon recognition by release factors is unknown and represents a long-standing coding problem of considerable interest. It entails protein-RNA recognition rather than the well understood mRNA-tRNA interaction in codon-anticodon pairing (2, 5, 6). The fact that two RFs from prokaryotes exhibit codon specificity suggests that they must interact directly with the codon. On accumulation of RF sequences from different organisms, the conservation of protein motifs has emerged in prokaryotic and eukaryotic RFs, as well as in the C-terminal portion of elongation factor EF-G, a translocase protein that forwards peptidyl tRNA from the A site to the P site on the ribosome (7). The three-dimensional structure of Thermus thermophilus EF-G comprises five subdomains; the C-terminal part, domains III-V, appears to mimic the shapes of the acceptor stem, the anticodon helix, and the T stem of tRNA, respectively (8-10). Furthermore, it appears that an RF region shares homology with domain IV of EF-G, thus constituting a putative ''tRNA-mimicry'' domain necessary for RF binding to the ribosomal A site (7). This mimicry model would explain why RFs recognize stop codons by assuming an anticodon- mimicry element in the protein and further suggest that all prokaryotic and eukaryotic RFs evolved from the progenitor of EF-G. RF1 and RF2 are known to be structurally similar, and both read the UAA codon. It might be possible, therefore, to alter mutationally either factor so that its stop codon specificity is altered. In the present study, we mutationally altered RF2 and show that a single amino acid substitution permits it to terminate translation at the UAG stop codon as well as the UGA and UAA stop codons, providing genetic support for the existence of the anticodon mimicry element in protein release factors.