Fakih, F.; Shulgina, Y.; Lukes, J.; Butenko, A.

The list of alternative genetic codes continues to grow, yet sense-to-sense codon reassignments remain very rare. Here we analyze a new sense-to-sense codon reassignment in bacteria, where the ACA codon, which canonically specifies threonine, has been predicted to encode aspartate. This so far unprecedented genetic code alteration is confined to a clade within the RAAP-2 family of the order Acidimicrobiales (phylum Actinomycetota). Validated by multiple alignments of highly conserved proteins, the reassignment is further supported by the presence of a tRNAUGU that lacks the G1:C72 threonylation identity element, suggesting that this tRNA is charged with aspartate instead of threonine. Our findings not only expand the set of known natural departures from the canonical genetic code by a novel type of sense-to-sense reassignment but also implicate the ACA codon that was so far considered invariably conserved in its meaning.