R213I mutation in release factor 2 (RF2) is one step forward for engineering an omnipotent release factor in bacteria Escherichia coli [RNA]

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 R213 as a crucial residue on Escherichia coli RF2 for discriminating guanine in the third position (G3). Interestingly, R213 is highly conserved in RF2 and substituted by I196 in the corresponding position in RF1. Another similar pair is L126 in RF1 and D143 in RF2, which are also conserved within their respective groups. With the hypothesis that replacement of R213 and D143 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 five-fold improved reading of the RF1-specific UAG codon relative to UAA, the universal stop codon, compared to the 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 SPT/PXT motifs. We propose that R213I mutation in RF2 brings us one step forward towards engineering an omnipotent RF in bacteria, capable of reading all three stop codons.

from #AlexandrosSfakianakis via Alexandros G.Sfakianakis on Inoreader http://ift.tt/2uuqMah
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