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

Gürkan Korkmaz,Suparna Sanyal

doi:10.1074/jbc.m117.785238

Abstract

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.

Highlights

Peptide release during translation termination is the end of protein synthesis on the ribosome
When RF1 and RF2 amino acid sequences from various bacteria are aligned together focusing mainly on the stop codon reading elements, a high degree of homology was observed between the two groups with some exceptions, which are moderately well conserved within the group
The tRNA mimicry hypothesis is certainly valid from the structural viewpoint, as the release factors (RFs) span from the decoding center to the peptidyl transferase center, like the tRNAs, and the tripeptide motifs PXT in RF1 and SPF in RF2 appear in the similar positions as the tRNA anticodons, but it oversimplifies the mechanism of stop codon recognition

Summary

To whom correspondence should be addressed

Class I release factors RF13 and RF2 recognize overlapping sets of three different termination signals (stop codons). Biochemical data are available for the alanine mutants of few conserved residues within the decoding region of RF1 (Gln-185, Arg-186, Thr-190, His-197, and Thr-198) [13, 14] These mutations showed a modest decrease in the maximal rate of peptide release (kcat) but resulted in a large 100-fold increase in the dissociation constant (kD) when bound to the ribosome with a cognate stop codon in the A site. Using site-directed mutagenesis, we generated several E. coli RF1/RF2 variants (RF1 I196A, RF1 I196R, RF1 L126D, RF2 R213A, RF2 R213I, and RF2 D143L) and characterized those in a competition assay for single-round peptide release [19] from ribosomal RCs containing major stop codon UAA (RCUAA) versus RCXXX carrying various codons (symbolized as XXX) in the A site These include UAG and UGA stop codons and UGG (Trp), UCA (Ser), and AAA (Lys) codons.

Results

Discussion

Experimental procedures