Structural and biochemical investigation of disease causing mutations in human mitochondrial tRNA Met

Mammalian mitochondria use a streamlined decoding mechanism requiring only 22 tRNAs, which are all encoded in the mitochondrial genome. The genes encoding mammalian mitochondrial tRNAs are hotspots for mutations leading to human disease. One tRNA with 3 known disease causing mutations is tRNA Met . To understand how each of these mutations affects the structure of tRNA Met we have used SHAPE chemistry to investigate the structure of the wild‐type and mutant tRNA transcripts. One tRNA Met mutation (U8C) results in growth retardation, dystrophic muscles and exercise intolerance in the afflicted patient. SHAPE analysis reveals that the wild‐type tRNA forms a cloverleaf structure similar to canonical tRNAs; however, the U8C mutation results in the loss of both the T and acceptor stems. The U at position 8 is unpaired but is a universally conserved nucleotide. In the absence of magnesium, wild‐type tRNA forms a structure similar to the U8C mutant. In contrast the absence of magnesium has little effect on the U8C mutant. These experiments suggest that the U8C mutation results in the loss of a magnesium binding site necessary for proper tRNA folding. As expected the U8C mutant does not aminoacylate well. A second mutation in tRNA Met (A37G) is located 3′ to the anticodon. This mutation increases the penetrance of a primary mutation causing Leber's hereditary optical neuropathy. SHAPE analysis reveals that this mutation has little effect on the structure of the tRNA, and the mutant tRNA aminoacylates well. This mutation may affect the ability of tRNA Met to decode. A mutation found in the T stem (G53A) results in abnormal mitochondria with defects in energy production. SHAPE reveals that this tRNA has weakened T and acceptor stems. The tRNA however does aminoacylate well.