Pathogenic
mechanism of a human mitochondrial tRNAPhe mutation associated with
myoclonic epilepsy with ragged red fibers syndrome.
Ohio State Biochemistry Program, Ohio State University, Columbus, OH 43210, USA.
Abstract
Human
mitochondrial tRNA (hmt-tRNA) mutations are associated with a variety
of diseases including mitochondrial myopathies, diabetes,
encephalopathies, and deafness. Because the current understanding of the
precise molecular mechanisms of these mutations is limited, there is no
efficient method to treat their associated mitochondrial diseases.
Here, we use a variety of known mutations in hmt-tRNA(Phe) to
investigate the mechanisms that lead to malfunctions. We tested the
impact of hmt-tRNA(Phe) mutations on aminoacylation, structure, and
translation elongation-factor binding. The majority of the mutants were
pleiotropic, exhibiting defects in aminoacylation, global structure, and
elongation-factor binding. One notable exception was the G34A anticodon
mutation of hmt-tRNA(Phe) (mitochondrial DNA mutation G611A), which is
associated with MERRF (myoclonic epilepsy with ragged red fibers). In
vitro, the G34A mutation decreases aminoacylation activity by 100-fold,
but does not affect global folding or recognition by elongation factor.
Furthermore, G34A hmt-tRNA(Phe) does not undergo adenosine-to-inosine
(A-to-I) editing, ruling out miscoding as a possible mechanism for
mitochondrial malfunction. To improve the aminoacylation state of the
mutant tRNA, we modified the tRNA binding domain of the nucleus-encoded
human mitochondrial phenylalanyl-tRNA synthetase, which aminoacylates
hmt-tRNA(Phe) with cognate phenylalanine. This variant enzyme displayed
significantly improved aminoacylation efficiency for the G34A mutant,
suggesting a general strategy to treat certain classes of mitochondrial
diseases by modification of the corresponding nuclear gene.
