The pathogenic A4269G mutation in human mitochondrial tRNAIle alters the T-stem structure and decreases the binding affinity for elongation factor Tu

The A4269G mutation in the human mitochondrial (mt) tRNA(Ile) gene is associated with fatal cardiomyopathy. This mutation completely inhibits protein synthesis in mitochondria, thereby significantly reducing their respiratory activity. The steady-state amount of tRNA(Ile) in cells bearing the A4269G mutation is almost half that of control cells. We previously reported that this mutation causes tRNA(Ile) to be unstable both in vivo and in vitro. To investigate whether the instability of the mutant tRNA(Ile) is due to structural alterations, a nuclease-probing experiment was performed with a mitochondrial enzymatic extract, which showed that the A4269G mutation destabilizes the T-stem of the mutant tRNA(Ile). In addition, measurements of the binding affinity of the aminoacylated mutant tRNA(Ile) for mt elongation factor Tu (EF-Tu) showed that the mutant tRNA(Ile) binds mt EF-Tu less efficiently than the wild-type does. This observation provides insight into the molecular pathology associated with tRNA dysfunction caused by this pathogenic point mutation.