Speaker
Description
Translation termination involves the recognition of stop codons by release factors (RFs). In bacteria, RF2 recognizes UGA and UAA stop codons and is encoded by the prfB gene. The translation of prfB is interrupted by an internal UGA stop codon on its coding sequence, resulting in negative autoregulation controlled by RF2 concentration. Complete translation can only occur when the internal stop codon is bypassed by a +1 programmed ribosomal frameshifting (PRF) event, which is more likely to occur when cellular RF2 level are low. In addition to RF2 concentration, frameshifting rate depends on conserved sequences near the internal stop codon, which interact with the ribosomal complex and facilitate frameshifting, thereby allowing sufficient expression of RF2. Although the PRF system is widely conserved across bacteria (PRF⁺ species), some bacterial species lack the internal stop codon in their prfB genes (PRF⁻ species) so that no frameshifting required while translation. It has been proposed that PRF loss may be beneficial in species with high UGA or UAA stop codon usage, but conclusive evidence is missing. In this study, we combine phylogenetic correlation analysis and experimental evolution to investigate the evolutionary pressures that lead to PRF loss. From phylogenetic analysis, loss of PRF did not correlate with increased usage of UGA or UAA stop codons, indicating that it is not driven by the genomic context. To further explore potential mechanisms of PRF loss, we constructed set of mutations in the sequences surrounding the internal stop codon of the prfB gene in Pseudomonas fluorescens SBW25 to disrupt its interaction with the ribosomal complex, leading to a reduced frameshifting rate. Using experimental evolution, we identified two compensatory strategies to overcome reduced frameshifting rate: (i) secondary mutations in ribosomal genes that can increase the frameshifting rate, and (ii) a single base pair deletion upstream of the internal stop codon, which shifts the reading frame and thereby bypasses the stop codon without recognition. These results demonstrate that the loss of PRF can occur as a consequence of compensatory evolution in response to deleterious mutations that reduce the frameshifting rate. This evolutionary trajectory aligns with the nearly neutral theory of molecular evolution, rather than with direct adaptive selection driven by stop codon usage.
