R213I mutation in release factor 2 (RF2) is one step forward for engineering an omnipotent release factor in bacteria...

The current understanding of the specificity of the bacterial class I release factors (RFs) in decoding stop codons has evolved beyond a simple tripeptide anticodon model. A recent molecular dynamics study for deciphering the principles for specific stop codon recognition by RFs identified Arg-213 as a crucial residue on Escherichia coli RF2 for discriminating guanine in the third position (G3). Interestingly, Arg-213 is highly conserved in RF2 and substituted by Ile-196 in the corresponding position in RF1. Another similar pair is Leu-126 in RF1 and Asp-143 in RF2, which are also conserved within their respective groups. With the hypothesis that replacement of Arg-213 and Asp-143 with the corresponding RF1 residues will reduce G3 discrimination by RF2, we swapped these residues between E. coli RF1 and RF2 by site-directed mutagenesis and characterized their preference for different codons using a competitive peptide release assay. Among these, the R213I mutant of RF2 showed 5-fold improved reading of the RF1-specific UAG codon relative to UAA, the universal stop codon, compared with the wild type (WT). In-depth fast kinetic studies revealed that the gain in UAG reading by RF2 R213I is associated with a reduced efficiency of termination on the cognate UAA codon. Our work highlights the notion that stop codon recognition involves complex interactions with multiple residues beyond the PXT/SPF motifs. We propose that the R213I mutation in RF2 brings us one step forward toward engineering an omnipotent RF in bacteria, capable of reading all three stop codons.