A T-stem slip in human mitochondrial tRNA(Leu)(CUN) governs its charging capacity

The human mitochondrial tRNA(Leu)(CUN) [hmtRNA(Leu)(CUN)] corresponds to the most abundant codon for leucine in human mitochondrial protein genes. Here, in vitro studies reveal that the U48C substitution in hmtRNA(Leu)(CUN), which corresponds to the pathological T12311C gene mutation, improved the aminoacylation efficiency of hmtRNA(Leu)(CUN). Enzymatic probing suggested a more flexible secondary structure in the wild-type hmtRNA(Leu)(CUN) transcript compared with the U48C mutant. Structural analysis revealed that the flexibility of hmtRNA(Leu)(CUN) facilitates a T-stem slip resulting in two potential tertiary structures. Several rationally designed tRNA(Leu)(CUN) mutants were generated to examine the structural and functional consequences of the T-stem slip. Examination of these hmtRNA(Leu)(CUN) mutants indicated that the T-stem slip governs tRNA accepting activity. These results suggest a novel, self-regulation mechanism of tRNA structure and function.