tRNA^Val allows four-way decoding with unmodified uridine at the wobble position in Lactobacillus casei

Modifications at the wobble position (position 34) of tRNA facilitate interactions that enable or stabilize non-Watson–Crick base pairs. In bacterial tRNA, 5-hydroxyuridine (ho⁵U) derivatives xo⁵U [x: methyl (mo⁵U), carboxymethyl (cmo⁵U), and methoxycarbonylmethyl (mcmo⁵U)] present at the wobble positions of tRNAs are responsible for the recognition of NYN codon families. These modifications of U34 allow base-pairing not only with A and G but also with U, and in some cases, C. mo⁵U was originally found in Gram-positive bacteria, and cmo⁵U and mcmo⁵U were found in Gram-negative bacteria. tRNAs of Mycoplasma species, mitochondria, and chloroplasts adopt four-way decoding in which unmodified U34 recognizes codons ending in A, G, C, and U. Lactobacillus casei, Gram-positive bacteria, and lactic acid bacteria lack the modification enzyme genes for xo⁵U biosynthesis. Nevertheless, L. casei has only one type of tRNA^Val with the anticodon UAC [tRNA^Val(UAC)]. However, the genome of L. casei encodes an undetermined tRNA (tRNA^Und) gene, and the sequence corresponding to the anticodon region is GAC. Here, we confirm that U34 in L. casei tRNA^Val is unmodified and that there is no tRNA^Und expression in the cells. In addition, in vitro transcribed tRNA^Und was not aminoacylated by L. casei valyl-tRNA synthetase, suggesting that tRNA^Und is not able to accept valine, even if expressed in cells. Correspondingly, native tRNA^Val(UAC) with unmodified U34 bound to all four valine codons in the ribosome A site. This suggests that L. casei tRNA^Val decodes all valine codons by four-way decoding, similarly to tRNAs from Mycoplasma species, mitochondria, and chloroplasts.