tRNA-dependent pre-transfer editing by prokaryotic leucyl-tRNA synthetase.
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.
Tan
Min
M
State Key Laboratory of Molecular Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Graduate School of the Chinese Academy of Sciences, The Chinese Academy of Sciences, Shanghai 200031, China.
Zhu
Bin
B
Zhou
Xiao-Long
XL
He
Ran
R
Chen
Xin
X
Eriani
Gilbert
G
Wang
En-Duo
ED
D000595:Amino Acid Sequence; D001419:Bacteria / Q000201:enzymology*; D001665:Binding Sites; D002384:Catalysis; D004926:Escherichia coli / Q000201:enzymology*; D007700:Kinetics; D007935:Leucine-tRNA Ligase / Q000737:chemistry*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D017393:RNA Editing; D012343:RNA, Transfer / Q000737:chemistry*; D012346:RNA, Transfer, Amino Acyl / Q000737:chemistry*; D017386:Sequence Homology, Amino Acid; D014364:Tryptophan / Q000737:chemistry
D012346:RNA, Transfer, Amino Acyl; D014364:Tryptophan; D012343:RNA, Transfer; D007935:Leucine-tRNA Ligase