Recognition of human mitochondrial tRNALeu(UUR) by its cognate leucyl-tRNA synthetase.
Accuracy of protein synthesis depends on specific recognition and aminoacylation of tRNAs by their cognate aminoacyl-tRNA synthetases. Rules governing these processes have been established for numerous prokaryotic and eukaryotic cytoplasmic systems, but only limited information is available for human mitochondrial systems. It has been shown that the in vitro transcribed human mitochondrial tRNA(Leu(UUR)) does not fold into the expected cloverleaf, but is however aminoacylated by the human mitochondrial leucyl-tRNA synthetase. Here, the role of the structure of the amino acid acceptor branch and the anticodon branch of tRNA(Leu(UUR)) in recognition by leucyl-tRNA synthetase was investigated. The kinetic parameters for aminoacylation of wild-type and mutant tRNA(Leu(UUR)) transcripts and of native tRNA(Leu(UUR)) were determined. Solution structure probing was performed in the presence or in the absence of leucyl-tRNA synthetase and correlated with the aminoacylation kinetics for each tRNA. Replacement of mismatches in either the anticodon-stem or D-stem that are present in the wild-type tRNA(Leu(UUR)) by G-C base-pairs is sufficient to induce (i) cloverleaf folding, (ii) improved aminoacylation efficiency, and (iii) interactions with the synthetase that are similar to those with the native tRNA(Leu(UUR)). Leucyl-tRNA synthetase contacts tRNA(Leu(UUR)) in the amino acid acceptor stem, the anticodon stem, and the D-loop, which is unprecedented for a leucine aminoacylation system.
Sohm
Bénédicte
B
UPR 9002 du CNRS, Institut de Biologie Moléculaire et Cellulaire, 15 rue René Descartes, 67084 Strasbourg Cedex, France.
Sissler
Marie
M
Park
Hyejeong
H
King
Michael P
MP
Florentz
Catherine
C
D000215:Acylation; D001483:Base Sequence; D006801:Humans; D007700:Kinetics; D007930:Leucine / Q000378:metabolism; D007935:Leucine-tRNA Ligase / Q000235:genetics / Q000378:metabolism*; D008928:Mitochondria / Q000378:metabolism*; D008969:Molecular Sequence Data; D009154:Mutation; D009690:Nucleic Acid Conformation; D012516:Osteosarcoma / Q000378:metabolism; D012313:RNA / Q000235:genetics* / Q000378:metabolism; D012356:RNA, Transfer, Leu / Q000235:genetics / Q000378:metabolism*; D012996:Solutions; D013379:Substrate Specificity; D014158:Transcription, Genetic / Q000235:genetics*; D014407:Tumor Cells, Cultured
D012356:RNA, Transfer, Leu; D012996:Solutions; D012313:RNA; D007935:Leucine-tRNA Ligase; D007930:Leucine