tRNA^Valallows four-way decoding with unmodified uridine at the wobble position in<i>Lactobacillus casei</i>

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 <i>Mycoplasma</i> species, mitochondria, and chloroplasts adopt four-way decoding in which unmodified U34 recognizes codons ending in A, G, C, and U. <i>Lactobacillus casei</i>, Gram-positive bacteria, and lactic acid bacteria lack the modification enzyme genes for xo⁵U biosynthesis. Nevertheless, <i>L. casei</i> has only one type of tRNA^Val with the anticodon UAC [tRNA^Val(UAC)]. However, the genome of <i>L. casei</i> encodes an undetermined tRNA (tRNA^Und) gene, and the sequence corresponding to the anticodon region is GAC. Here, we confirm that U34 in <i>L. casei</i> 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 <i>L. casei</i> 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 <i>L. casei</i> tRNA^Val decodes all valine codons by four-way decoding, similarly to tRNAs from <i>Mycoplasma</i> species, mitochondria, and chloroplasts.