Transfer RNA modulates the editing mechanism used by class II prolyl-tRNA synthetase

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-tRNAPro. 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-tRNAPro formation in the presence of tRNAPro 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.