title: Pathogenic mechanism of a human mitochondrial tRNAPhe mutation associated with myoclonic epilepsy with ragged red fibers syndrome.
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.
authors: Ohio State Biochemistry Program, Ohio State University, Columbus, OH 43210, USA.
authors: Jiqiang
authors: J
authors: Ling
authors: Hervé
authors: H
authors: Roy
authors: Daoming
authors: D
authors: Qin
authors: Mary Anne T
authors: MA
authors: Rubio
authors: Juan D
authors: JD
authors: Alfonzo
authors: Kurt
authors: K
authors: Fredrick
authors: Michael
authors: M
authors: Ibba
mesh_terms: D000926:Anticodon; D001483:Base Sequence; D004831:Epilepsies, Myoclonic / Q000150:complications* / Q000235:genetics*; D006801:Humans; D007700:Kinetics; D017243:MERRF Syndrome / Q000150:complications* / Q000235:genetics*; D008928:Mitochondria / Q000378:metabolism; D008968:Molecular Conformation; D008969:Molecular Sequence Data; D009154:Mutation; D009690:Nucleic Acid Conformation; D010444:Peptide Elongation Factor Tu / Q000378:metabolism; D010649:Phenylalanine / Q000737:chemistry; D012313:RNA / Q000737:chemistry*; D000077278:RNA, Mitochondrial; D012360:RNA, Transfer, Phe / Q000737:chemistry / Q000235:genetics*
chemical_list: D000926:Anticodon; D000077278:RNA, Mitochondrial; D012360:RNA, Transfer, Phe; D010649:Phenylalanine; D012313:RNA; D010444:Peptide Elongation Factor Tu