Biogenesis of glutaminyl-mt tRNAGln in human mitochondria.
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.
Nagao
Asuteka
A
Department of Chemistry and Biotechnology, Graduate School of Engineering, University of Tokyo, Building 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan.
Suzuki
Takeo
T
Katoh
Takayuki
T
Sakaguchi
Yuriko
Y
Suzuki
Tsutomu
T
D000595:Amino Acid Sequence; D000604:Amino Acyl-tRNA Synthetases / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D015152:Blotting, Northern; D002417:Cattle; D005975:Glutamate-tRNA Ligase / Q000235:genetics / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D007700:Kinetics; D008856:Microscopy, Fluorescence; D008928:Mitochondria / Q000378:metabolism*; D008969:Molecular Sequence Data; D019884:Nitrogenous Group Transferases / Q000235:genetics / Q000378:metabolism; D009690:Nucleic Acid Conformation; D021122:Protein Subunits / Q000235:genetics / Q000378:metabolism; D034622:RNA Interference; D012346:RNA, Transfer, Amino Acyl / Q000096:biosynthesis* / Q000737:chemistry; D012351:RNA, Transfer, Gln / Q000096:biosynthesis* / Q000737:chemistry; D012352:RNA, Transfer, Glu / Q000096:biosynthesis / Q000737:chemistry; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D014162:Transfection; D046249:Transfer RNA Aminoacylation
D021122:Protein Subunits; D012346:RNA, Transfer, Amino Acyl; D012351:RNA, Transfer, Gln; D012352:RNA, Transfer, Glu; D011993:Recombinant Fusion Proteins; C483187:glutamyl-tRNA(Gln); D049452:Green Fluorescent Proteins; D019884:Nitrogenous Group Transferases; D000604:Amino Acyl-tRNA Synthetases; D005975:Glutamate-tRNA Ligase; C020659:glutaminyl-tRNA synthetase