AUA codon decoding by preferential use of tRNA Ile (UAU) in Lactobacillus casei

Modified nucleosides at the first (wobble) position of tRNA anticodons play critical roles in accurate decoding of the genetic code. In bacteria, the isoleucine AUA codon is typically decoded by tRNA Ile (LAU), in which lysidine (L) at the wobble position of tRNA Ile with a CAU anticodon ensures discrimination from the methionine AUG codon. However, some bacteria, such as Mycoplasma mobile , lack tRNA Ile (LAU) and instead utilize tRNA Ile (UAU). In this organism, the unmodified uridine at the wobble position is thought to enable specific decoding of AUA while avoiding AUG recognition. In our previous study, we identified a lactic acid bacterium in which both tRNA Ile (LAU) and tRNA Ile (UAU) coexist. Here, we show that tRNA Ile (LAU) is scarcely aminoacylated in vivo , whereas tRNA Ile (UAU) is efficiently aminoacylated. Notably, the presence of 4-thiouridine (s 4 U) at position 8 inhibits IleRS-dependent aminoacylation of tRNA Ile (UAU) in vitro , suggesting a regulatory role of tRNA modification in this process. Moreover, tRNA Ile (LAU) exhibits incomplete discrimination between AUA and AUG codons and binds to AUG in the ribosomal A-site binding assays. In contrast, tRNA Ile (UAU) containing N 6 -threonylcarbamoyladenosine (t 6 A) at position 37 showed a tendency toward improved discrimination between AUA and AUG codons and preferentially recognized AUA at the ribosomal A site. These results indicate that AUA decoding is predominantly mediated by preferential use of tRNA Ile (UAU) rather than canonical tRNA Ile (LAU), revealing an alternative mechanism of codon decoding based on differential utilization of tRNA isoacceptors, and providing an additional layer of translational control in bacteria.