Tan, M, Zhu, B, Zhou, XL, He, R, Chen, X, Eriani, G and Wang, ED (2010) tRNA-dependent pre-transfer editing by prokaryotic leucyl-tRNA synthetase. J. Biol. Chem. 285:3235-44
To prevent genetic code ambiguity due to misincorporation of amino acids into proteins, aminoacyl-tRNA synthetases have evolved editing activities to eliminate intermediate or final non-cognate products. In this work we studied the different editing pathways of class Ia leucyl-tRNA synthetase (LeuRS). Different mutations and experimental conditions were used to decipher the editing mechanism, including the recently developed compound AN2690 that targets the post-transfer editing site of LeuRS. The study emphasizes the crucial importance of tRNA for the pre- and post-transfer editing catalysis. Both reactions have comparable efficiencies in prokaryotic Aquifex aeolicus and Escherichia coli LeuRSs, although the E. coli enzyme favors post-transfer editing, whereas the A. aeolicus enzyme favors pre-transfer editing. Our results also indicate that the entry of the CCA-acceptor end of tRNA in the editing domain is strictly required for tRNA-dependent pre-transfer editing. Surprisingly, this editing reaction was resistant to AN2690, which inactivates the enzyme by forming a covalent adduct with tRNA(Leu) in the post-transfer editing site. Taken together, these data suggest that the binding of tRNA in the post-transfer editing conformation confers to the enzyme the capacity for pre-transfer editing catalysis, regardless of its capacity to catalyze post-transfer editing.
Amino Acid Sequence; Bacteria/enzymology; Binding Sites; Catalysis; Escherichia coli/enzymology; Kinetics; Leucine-tRNA Ligase/chemistry; Models, Molecular; Molecular Sequence Data; Mutation; RNA Editing; RNA, Transfer/chemistry; RNA, Transfer, Amino Acyl/chemistry; Sequence Homology, Amino Acid; Tryptophan/chemistry
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