The A4269G mutation in the human mitochondrial (mt) tRNAIle 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 tRNAIle in cells bearing the A4269G mutation is almost half that of control cells. We previously reported that this mutation causes tRNAIle to be unstable both in vivo and in vitro. To investigate whether the instability of the mutant tRNAIle 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 tRNAIle. In addition, measurements of the binding affinity of the aminoacylated mutant tRNAIle for mt elongation factor Tu (EF-Tu) showed that the mutant tRNAIle 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.
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