The pathogenic A4269G mutation in human mitochondrial tRNA ^Ile 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.