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J. Biol. Chem., Vol. 281, Issue 38, 27862-27872, September 22, 2006
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From the
Departments of Chemistry and Biochemistry, Molecular Biology and Biophysics, University of Minnesota, Minnesota 55455 and the ¶Department of Chemistry, Carleton College, Northfield, Minnesota 55057
Aminoacyl-tRNA synthetases catalyze the attachment of cognate amino acids to specific tRNA molecules. To prevent potential errors in protein synthesis caused by misactivation of noncognate amino acids, some synthetases have evolved editing mechanisms to hydrolyze misactivated amino acids (pre-transfer editing) or misacylated tRNAs (post-transfer editing). In the case of post-transfer editing, synthetases employ a separate editing domain that is distinct from the site of amino acid activation, and the mechanism is believed to involve shuttling of the flexible CCA-3' end of the tRNA from the synthetic active site to the site of hydrolysis. The mechanism of pre-transfer editing is less well understood, and in most cases, the exact site of pre-transfer editing has not been conclusively identified. Here, we probe the pre-transfer editing activity of class II prolyl-tRNA synthetases from five species representing all three kingdoms of life. To locate the site of pre-transfer editing, truncation mutants were constructed by deleting the insertion domain characteristic of bacterial prolyl-tRNA synthetase species, which is the site of post-transfer editing, or the N- or C-terminal extension domains of eukaryotic and archaeal enzymes. In addition, the pre-transfer editing mechanism of Escherichia coli prolyl-tRNA synthetase was probed in detail. These studies show that a separate editing domain is not required for pre-transfer editing by prolyl-tRNA synthetase. The aminoacylation active site plays a significant role in preserving the fidelity of translation by acting as a filter that selectively releases non-cognate adenylates into solution, while protecting the cognate adenylate from hydrolysis.
Received for publication, June 19, 2006 , and in revised form, July 18, 2006.
* This work was supported by National Institutes of Health Grant GM49928 (to K. M.-F.) and a grant from the Arnold and Mabel Beckman Foundation (to B. Z.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1 Both authors contributed equally to this work.
2 To whom correspondence should be addressed: Dept. of Chemistry, University of Minnesota, 207 Pleasant St. SE, Minneapolis, MN 55455. Tel.: 612-624-0286; Fax: 612-626-7541; E-mail: musier{at}chem.umn.edu.
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