We present a comprehensive analysis of stop codon usage in bacteria by analyzing over eight million coding sequences of 4684 bacterial sequences. Using a newly developed program called “stop codon counter,” the frequencies of the three classical stop codons TAA, TAG, and TGA were analyzed, and a publicly available stop codon database was built. Our analysis shows that with increasing genomic GC content the frequency of the TAA codon decreases and that of the TGA codon increases in a reciprocal manner. Interestingly, the release factor 1-specific codon TAG maintains a more or less uniform frequency (∼20%) irrespective of the GC content. The low abundance of TAG is also valid with respect to expression level of the genes ending with different stop codons. In contrast, the highly expressed genes predominantly end with TAA, ensuring termination with either of the two release factors. Using three model bacteria with different stop codon usage (Escherichia coli, Mycobacterium smegmatis, and Bacillus subtilis), we show that the frequency of TAG and TGA codons correlates well with the relative steady state amount of mRNA and protein for release factors RF1 and RF2 during exponential growth. Furthermore, using available microarray data for gene expression, we show that in both fast growing and contrasting biofilm formation conditions, the relative level of RF1 is nicely correlated with the expression level of the genes ending with TAG.

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.

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.

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 ( k _cat ) and Michaelis-Menten constant ( K _M ). 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. Abbreviations: RF release factor RC release complex