Biogenesis of glutaminyl-mt [tRNA.sup.Gln] 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 (GInRS) has been identified in mammalian genomes, implying that GIn-[tRNA.sup.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.sup.Gln] to form Glu-[tRNA.sup.GIn]. In addition, we have identified a human homolog of the Glu-[tRNA.sup.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.sup.Gln] accumulated and defects in respiration could be observed. We successfully reconstituted in vitro Gln-[tRNA.sup.Gln] formation catalyzed by the recombinant mtGluRS and hGatCAB. The misaminoacylated form of [tRNA.sup.Gln] has a weak binding affinity to the mt elongation factor Tu (mtEF-Tu), indicating that the misaminoacylated form of [tRNA.sup.Gln] is rejected from the translational apparatus to maintain the accuracy of mitochondrial protein synthesis. EF-Tu | GIu-tRNAGln amidotransferase | glutamyl-tRNA synthetase | mitochondrial tRNA