Background: Stop codon frequencies in 4684 bacterial genomes are analyzed. Results: With increasing genomic GC content, TAA% decreases, and TGA% increases reciprocally, but TAG% remains almost unchanged (ϳ20%). The TAG:TGA ratio matches well with the RF1:RF2 ratio. Conclusion: TAG is the minor stop codon, and expression of genes with TAG is correlated with the RF1 level. Significance: Our work establishes the correlation between stop codon frequency and RF1/RF2 abundance.

Termination of messenger RNA translation in Bacteria and Archaea is initiated by release factors (RFs) 1 or 2 recognizing a stop codon in the ribosomal A site and releasing the peptide from the P-site transfer RNA. After release, RF-dissociation is facilitated by the G-protein RF3. Structures of ribosomal complexes with RF1 or RF2 alone or with RF3 alone—RF3 bound to a non-hydrolyzable GTP-analog—have been reported. Here, we present the cryo-EM structure of a post-termination ribosome containing both apo-RF3 and RF1. The conformation of RF3 is distinct from those of free RF3•GDP and ribosome-bound RF3•GDP(C/N)P. Furthermore, the conformation of RF1 differs from those observed in RF3-lacking ribosomal complexes. Our study provides structural keys to the mechanism of guanine nucleotide exchange on RF3 and to an L12-mediated ribosomal recruitment of RF3. In conjunction with previous observations, our data provide the foundation to structurally characterize the complete action cycle of the G-protein RF3.DOI: http://dx.doi.org/10.7554/eLife.00411.001

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 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 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 PT/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.

Translation termination is the last step of protein synthesis where the newly synthesized protein is released from the ribosome. For this, a class I release factor (RF1 and RF2) in bacteria binds to one of the three stop codons (UAA, UAG and UGA) and hydrolyze the ester bond between nascent protein and the peptidyl‐tRNA. RF1 and RF2, although similar to each other, read the stop codons in a semi specific manner i.e. RF1 reads UAA and UAG, and RF2 reads UAA and UGA codons. According to current literature [1] these factors discriminate highly against sense codons. Although molecular dynamics simulation experiments [2] have proposed a network of interactions through which RF1 and RF2 recognizes their respective stop codons, there exist very little biochemical experiments to explain how this high specificity is achieved. Using site directed mutagenesis we generated several variants of RF1 and RF2 and characterized those in competition and kinetic assays. One of our engineered release factors showed omnipotent property in stop codon recognition. We have characterized this engineered release factor with respect to other related codons especially in comparison with other release factors. Further we were able to validate our biochemical experiments using computer simulations. Our experiments pinpointed the crucial residues on the class‐I release factors for stop codon specificity and enabled us to engineer a class I release factor capable of reading all three stop codons. Grant Funding Source: Supported by; Swedish Research Council, Sven and Lilly Lawski Foundation

Termination of messenger RNA translation in Bacteria and Archaea is initiated by release factors (RFs) 1 or 2 recognizing a stop codon in the ribosomal A site and releasing the peptide from the P-site transfer RNA. After release, RF-dissociation is facilitated by the G-protein RF3. Structures of ribosomal complexes with RF1 or RF2 alone or with RF3 alone-RF3 bound to a non-hydrolyzable GTP-analog-have been reported. Here, we present the cryo-EM structure of a post-termination ribosome containing both apo-RF3 and RF1. The conformation of RF3 is distinct from those of free RF3•GDP and ribosome-bound RF3•GDP(C/N)P. Furthermore, the conformation of RF1 differs from those observed in RF3-lacking ribosomal complexes. Our study provides structural keys to the mechanism of guanine nucleotide exchange on RF3 and to an L12-mediated ribosomal recruitment of RF3. In conjunction with previous observations, our data provide the foundation to structurally characterize the complete action cycle of the G-protein RF3.

Ribosomal protein synthesis (translation) is a highly accurate process. Translation termination, in particular, must be accurate to prevent truncated proteins. How this accuracy is achieved is not fully understood in all its details. Using an E. coli in vitro system, I explore novel mechanisms that contribute to the high accuracy of translation termination. By comparing the Michaelis-Menten parameters of methylated and non-methylated release factors on cognate and non-cognate codons. Post-translational methylation of a strictly conserved GGQ motif in class I release factors increases the accuracy of termination by up to 5-fold. This happens by increasing both the maximum rate of peptide release (kcat) and Michaelis-Menten constant (KM). Further, I demonstrate here that a non-methylated release factor acts like an uncompetitive inhibitor of enzyme reactions. Overall, this study shows that the methylation of class I release factors is a novel mechanism contributing to highly accurate translation termination.AbbreviationsRFrelease factorRCrelease complex