tRNA allows four-way decoding with unmodified uridine at the wobble position in .

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 (hoU) derivatives xoU [x: methyl (moU), carboxymethyl (cmoU), and methoxycarbonylmethyl (mcmoU)] 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. moU was originally found in Gram-positive bacteria, and cmoU and mcmoU were found in Gram-negative bacteria. tRNAs of species, mitochondria, and chloroplasts adopt four-way decoding in which unmodified U34 recognizes codons ending in A, G, C, and U. , Gram-positive bacteria, and lactic acid bacteria lack the modification enzyme genes for xoU biosynthesis. Nevertheless, has only one type of tRNA with the anticodon UAC [tRNA(UAC)]. However, the genome of encodes an undetermined tRNA (tRNA) gene, and the sequence corresponding to the anticodon region is GAC. Here, we confirm that U34 in tRNA is unmodified and that there is no tRNA expression in the cells. In addition, in vitro transcribed tRNA was not aminoacylated by valyl-tRNA synthetase, suggesting that tRNA is not able to accept valine, even if expressed in cells. Correspondingly, native tRNA(UAC) with unmodified U34 bound to all four valine codons in the ribosome A site. This suggests that tRNA decodes all valine codons by four-way decoding, similarly to tRNAs from species, mitochondria, and chloroplasts.