Conservation and evolution of the programmed ribosomal frameshift in prfB across the bacterial domain

When the ribosome reaches a stop codon, translation is terminated by a release factor. Bacteria encode two release factors, RF1 and RF2. In many bacteria, the gene encoding RF2 ( prfB) contains an in-frame premature stop codon near the beginning of the open reading frame. A programmed ribosomal frameshift is therefore required to translate full-length RF2. While the molecular mechanism of the programmed ribosomal frameshift has been extensively characterized in Escherichia coli , bioinformatic analysis of the evolution and conservation of this motif has been limited to few genomes. By analyzing >12,000 bacterial genomes, we sought to thoroughly characterize the conserved frameshifting elements within the programmed frameshifting motif and identify genomic features of phyla that have lost the motif altogether. We find that the programmed ribosomal frameshift in prfB was likely present in the last common ancestor of bacteria and that the motif elements are almost completely conserved, including the identity of the internal stop codon. We find that loss of the programmed frameshift motif is highly correlated with RF2-specific stop codon usage, suggesting that stop codon usage has shaped the conservation of this regulatory mechanism. In support of this model, the programmed frameshift in prfB is entirely absent in Actinobacteriota, which have particularly high RF2 specific stop codon usage. Finally, we show that a model member of Actinobacteriota fails to produce full-length RF2 when provided with an allele of prfB that contains the programmed frameshifting motif. Altogether, our work provides a thorough characterization of RF2 regulation across the bacterial domain.