tRNA-dependent Pre-transfer Editing by Prokaryotic Leucyl-tRNA Synthetase*

  1. En-Duo Wang‡,2
  1. From the ‡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, 320 Yue Yang Road, Shanghai 200031, China and
  2. §Architecture et Réactivité de l'ARN, UPR9002 du CNRS, Institut de Biologie Moléculaire et Cellulaire, Université de Strasbourg, 15 rue René Descartes, 67084 Strasbourg, France
  1. 2 To whom correspondence should be addressed. Tel.: 86-21-5492-1241; Fax: 86-21-5492-1011; E-mail: edwang{at}sibs.ac.cn.

  1. ↵1 Both authors contributed equally to the work.

Abstract

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 tRNALeu 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.

Footnotes

  • ↵* This work was supported by Natural Science Foundation of China Grants 30670463 and 30930022, National Key Basic Research Foundation of China Grant 2006CB910301, the 973 Project of China Grant 2005CB724600, Committee of Science and Technology in Shanghai Grant 09JC1415900, and the Exchange Program and Programme International de Coopération Scientifique from CNRS Grant 3606.

  • ↵Graphic The on-line version of this article (available at http://www.jbc.org) contains supplemental Table 1 and Figs. S1 and S2.

  • ↵3 The abbreviations used are:

    aaRS
    aminoacyl-tRNA synthetase
    LeuRS
    leucyl-tRNA synthetase
    EcLeuRS
    E. coli LeuRS
    AaLeuRS
    A. aeolicus LeuRS
    Nva
    norvaline
    CP1
    connective peptide 1.

    • Received August 27, 2009.
    • Revision received November 20, 2009.
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  1. First Published on November 23, 2009 doi: 10.1074/jbc.M109.060616 The Journal of Biological Chemistry 285, 3235-3244.
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