Abstract
Aminoacyl-tRNA synthetases catalyze the attachment of amino acids to their cognate tRNAs. To prevent errors in protein synthesis, many synthetases have evolved editing pathways by which misactivated amino acids (pre-transfer editing) and misacylated tRNAs (post-transfer editing) are hydrolyzed. Previous studies have shown that class II prolyl-tRNA synthetase (ProRS) possesses both pre- and post-transfer editing functions against noncognate alanine. To assess the relative contributions of pre- and post-transfer editing, presented herein are kinetic studies of an Escherichia coli ProRS mutant in which post-transfer editing is selectively inactivated, effectively isolating the pre-transfer editing pathway. When post-transfer editing is abolished, substantial levels of alanine mischarging are observed under saturating amino acid conditions, indicating that pre-transfer editing alone cannot prevent the formation of Ala-tRNA Pro. Steady-state kinetic parameters for aminoacylation measured under these conditions reveal that the preference for proline over alanine is 2000-fold, which is well within the regime where editing is required. Simultaneous measurement of AMP and Ala-tRNA Pro formation in the presence of tRNA Pro suggested that misactivated alanine is efficiently transferred to tRNA to form the mischarged product. In the absence of tRNA, enzyme-catalyzed Ala-AMP hydrolysis is the dominant form of editing, with "selective release" of noncognate adenylate from the active site constituting a minor pathway. Studies with human and Methanococcus jannaschii ProRS, which lack a post-transfer editing domain, suggest that enzymatic pre-transfer editing occurs within the aminoacylation active site. Taken together, the results reported herein illustrate how both pre- and post-transfer editing pathways work in concert to ensure accurate aminoacylation by ProRS.
Highlights
Initial steady-state charging assays performed with proline revealed that both WT and K279A Prolyl-tRNA synthetase (ProRS) charge the cognate amino acid substrate with equal efficiency, indicating that the fidelity of the synthetic active site in this mutant is maintained
The presence of multiple editing pathways (Scheme 1) among the AARS highlights the importance of editing mechanisms in the fidelity of protein synthesis [33]
Class I MetRS is capable of catalyzing the cyclization of the homocysteine adenylate to form a thiolactone intermediate, representing a form of pre-transfer editing against this amino acid [36, 37]
Summary
Enzyme Preparation—Wild type (WT) and K279A Ec ProRS [12, 20], Hm ProRS [21], and Mj ProRS [22] were prepared as described previously. A 100-l reaction containing 50 mM glycineHCl buffer, pH 9.0, 10 mM MgCl2, 1 M tRNAPro, 0.82 M [␣-32P]ATP (Amersham Biosciences), 50 M sodium PPi, and 30 g/ml tRNA-terminal nucleotidyl transferase was incubated for 5 min at 37 °C. Enzyme Assays—Aminoacylation assays to determine kinetic parameters and charging levels were performed at 37 °C. TLC assays to measure the rate of AMP formation were performed as described previously [14], with the exception that pyrophosphatase was omitted from the reaction. The data were analyzed as described previously at 0.5 M enzyme concentrations [14]. The parameters were determined at pH 7.5 via Lineweaver-Burk analysis in the presence of varying amino acid concentrations, as described under “Experimental Procedures.”
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