Abstract
Mammalian mitochondrial (mt) tRNAs, which are required for mitochondrial protein synthesis, are all encoded in the mitochondrial genome, while mt aminoacyl-tRNA synthetases (aaRSs) are encoded in the nuclear genome. However, no mitochondrial homolog of glutaminyl-tRNA synthetase (GlnRS) has been identified in mammalian genomes, implying that Gln-tRNA(Gln) is synthesized via an indirect pathway in the mammalian mitochondria. We demonstrate here that human mt glutamyl-tRNA synthetase (mtGluRS) efficiently misaminoacylates mt tRNA(Gln) to form Glu-tRNA(Gln). In addition, we have identified a human homolog of the Glu-tRNA(Gln) amidotransferase, the hGatCAB heterotrimer. When any of the hGatCAB subunits were inactivated by siRNA-mediated knock down in human cells, the Glu-charged form of tRNA(Gln) accumulated and defects in respiration could be observed. We successfully reconstituted in vitro Gln-tRNA(Gln) formation catalyzed by the recombinant mtGluRS and hGatCAB. The misaminoacylated form of tRNA(Gln) has a weak binding affinity to the mt elongation factor Tu (mtEF-Tu), indicating that the misaminoacylated form of tRNA(Gln) is rejected from the translational apparatus to maintain the accuracy of mitochondrial protein synthesis.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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Amino Acid Sequence
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Amino Acyl-tRNA Synthetases / genetics
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Amino Acyl-tRNA Synthetases / metabolism*
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Animals
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Blotting, Northern
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Cattle
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Glutamate-tRNA Ligase / genetics
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Glutamate-tRNA Ligase / metabolism
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Green Fluorescent Proteins / genetics
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Green Fluorescent Proteins / metabolism
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HeLa Cells
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Humans
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Kinetics
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Microscopy, Fluorescence
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Mitochondria / metabolism*
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Molecular Sequence Data
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Nitrogenous Group Transferases / genetics
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Nitrogenous Group Transferases / metabolism
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Nucleic Acid Conformation
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Protein Subunits / genetics
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Protein Subunits / metabolism
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RNA Interference
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RNA, Transfer, Amino Acyl / biosynthesis*
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RNA, Transfer, Amino Acyl / chemistry
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RNA, Transfer, Gln / biosynthesis*
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RNA, Transfer, Gln / chemistry
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RNA, Transfer, Glu / biosynthesis
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RNA, Transfer, Glu / chemistry
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Recombinant Fusion Proteins / genetics
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Recombinant Fusion Proteins / metabolism
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Sequence Homology, Amino Acid
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Transfection
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Transfer RNA Aminoacylation
Substances
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Protein Subunits
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RNA, Transfer, Amino Acyl
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RNA, Transfer, Gln
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RNA, Transfer, Glu
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Recombinant Fusion Proteins
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glutamyl-tRNA(Gln)
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Green Fluorescent Proteins
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Nitrogenous Group Transferases
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Amino Acyl-tRNA Synthetases
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Glutamate-tRNA Ligase
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glutaminyl-tRNA synthetase