Post-transcriptional modifications at the first (wobble) position of the tRNA anticodon participate in precise decoding of the genetic code. To decode codons that end in a purine (R) (i.e. NNR), tRNAs frequently utilize 5-methyluridine derivatives (xm⁵U) at the wobble position. However, the functional properties of the C5-substituents of xm⁵U in codon recognition remain elusive. We previously found that mitochondrial tRNAs^Leu(UUR) with pathogenic point mutations isolated from MELAS (mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes) patients lacked the 5-taurinomethyluridine (τm⁵U) modification and caused a decoding defect. Here, we constructed Escherichia coli tRNAs^Leu(UUR) with or without xm⁵U modifications at the wobble position and measured their decoding activities in an in vitro translation as well as by A-site tRNA binding. In addition, the decoding properties of tRNA^Arg lacking mnm⁵U modification in a knock-out strain of the modifying enzyme (ΔmnmE) were examined by pulse labeling using reporter constructs with consecutive AGR codons. Our results demonstrate that the xm⁵U modification plays a critical role in decoding NNG codons by stabilizing U·G pairing at the wobble position. Crystal structures of an anticodon stem-loop containing τm⁵U interacting with a UUA or UUG codon at the ribosomal A-site revealed that the τm⁵U·G base pair does not have classical U·G wobble geometry. These structures provide help to explain how the τm⁵U modification enables efficient decoding of UUG codons.

Conventional tRNAs have highly conserved sequences, four-armed cloverleaf secondary structures, and L-shaped tertiary structures. However, metazoan mitochondrial tRNAs contain several exceptional structures. Almost all tRNASers for AGY/N codons lack the D-arm. Furthermore, in some nematodes, no four-armed cloverleaf-type tRNAs are present: two tRNAsSer without the D-arm and 20 tRNAs without the T-arm are found. Previously, we showed that in nematode mitochondria, an extra elongation factor Tu (EF-Tu) has evolved to support interaction with tRNAs lacking the T-arm, which interact with C-terminal domain 3 in conventional EF-Tu. Recent mitochondrial genome analyses have suggested that in metazoan lineages other than nematodes, tRNAs without the T-arm are present. Furthermore, even more simplified tRNAs are predicted in some lineages. In this review, we discuss mitochondrial tRNAs with divergent structures, as well as protein factors, including EF-Tu, that support the function of truncated metazoan mitochondrial tRNAs.

An unnatural base pair of 2-amino-6-( N , N -dimethylamino)purine (designated as x) and pyridin-2-one (designated as y) has been developed for specific transcription. The ribonucleoside triphosphates of y and a modified y, 5-methylpyridin-2-one, are selectively incorporated into RNA opposite x in the templates by T7 RNA polymerase. In addition, the sequences of the DNA templates containing x can be confirmed by a dideoxynucleotide chain-terminator method supplemented with the deoxynucleoside triphosphate of y. The bulky dimethylamino group of x in the templates effectively eliminates noncognate pairing with the natural bases. These results enable RNA biosynthesis for the specific incorporation of unnatural nucleotides at the desired positions.