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-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.
Splan
Kathryn E
KE
Department of Chemistry, University of Minnesota, Minneapolis, Minnesota 55455, USA.
Ignatov
Michael E
ME
Musier-Forsyth
Karin
K
D000255:Adenosine Triphosphate / Q000378:metabolism; D000409:Alanine / Q000737:chemistry; D000604:Amino Acyl-tRNA Synthetases / Q000737:chemistry*; D001665:Binding Sites; D004926:Escherichia coli / Q000201:enzymology; D006801:Humans; D006868:Hydrolysis; D007700:Kinetics; D017017:Methanococcus / Q000201:enzymology; D008954:Models, Biological; D008956:Models, Chemical; D009690:Nucleic Acid Conformation; D017393:RNA Editing; D012343:RNA, Transfer / Q000378:metabolism*; D013997:Time Factors
D000255:Adenosine Triphosphate; D012343:RNA, Transfer; D000604:Amino Acyl-tRNA Synthetases; C100134:prolyl T RNA synthetase; D000409:Alanine