Post-transcriptional modification at the first (wobble) position of the tRNA anticodon participates in precise decoding of the genetic code. We recently identified a novel taurine-containing modified uridine (tau m5U; 5-taurinomethyluridine) at the wobble position of mammalian mitochondrial tRNAs and found lack of this modification in mutant mitochondrial tRNAs from human pathogenic cells of the mitochondrial encephalomyopathies, investigate molecular pathogenesis of the diseases, decoding activity of wobble uridines with or without C5 modification was measured using E. coli cell-free translation system. It has been revealed that C5 modification has a functional role for stabilizing U:G wobble base pair.