Evolution And Mechanism Of Leucyl-tRNA Synthetase Editing Function

Leucyl-, isoleucyl- and valyl-tRNA synthetases form a subgroup of related aminoacyl-tRNA synthetases that catalyze the attachment of similar non-polar amino acids onto their cognate tRNAs. To maintain the fidelity of protein biosynthesis these enzymes also hydrolyze mischarged tRNAs through a post-transfer editing mechanism. Here we show that the ancestralαβ-LeuRS from Aquifex aeolicus and its isolated editing domain (AaLeu-CP1) catalyze the hydrolytic editing of the complete set of aminoacylated tRNAs generated by the three enzymes: Ile-tRNAIle, Val-tRNAIle, Val-tRNAVal, Thr-tRNAVal and Ile-tRNALeu indicating they have nonspecific editing function. A composite minihelixLIV was designed to carry the triple amino acid identity and to mimic the RNA molecule charged by the common ancestor of Leu-, Ile- and ValRS. We found that the three enzymes can aminoacylate minihelixLIV, indicating that they carry remnant-charging activity for this ancient form of transfer RNA. We tested if isolated CP1 domains could hydrolyze these primitive aminoacylated RNAs. We found that only the primitive AaLeu-CP1 could efficiently hydrolyze Ile-, Val- and Thr-minihelixLIV carrying non-cognate amino acids. These data indicate thatA?aLeu-CP1 has retained the hydrolytic function to edit the mischarged RNAs issued from the ancestor of Leu-, Ile- and ValRS. These results support the hypothesis that A. aeolicus -LeuRS is closer than Ile-, and ValRS from the last common ancestor from which it still carries the ambiguous editing function.Leucyl-tRNA synthetase (LeuRS) has editing functions to eliminate misactivated amino acids and mischarged tRNAs for preventing genetic code ambiguity, although the mechanisms are unclear. We studied the LeuRSs from Aquifex aeolicus, Escherichia coli, Pyrococcus horikoshii, Saccharomyces cerevisiae, human cytosol and human mitochondria and separated the total editing of LeuRS into three parts: tRNA-independent pretransfer editing, tRNA-dependent pretransfer editing, and posttransfer editing. The eukaryotic LeuRSs have no obvious tRNA-dependent pretransfer editing. The active site for tRNA-independent pretransfer editing appears to be the aminoacylation site.Leucyl-tRNA synthetase (LeuRS) was believed to have tRNA-dependent editing functions to eliminate misactivated amino acids (pretransfer editing) and mischarged tRNAs (post-transferring editing) for preventing genetic code ambiguity. Although the posttransfer editing had been well characterized, the pretransfer editing remained unclear due to the instability of its substrates and the overlapping of its active site with that of posttransfer editing. In the study, with the comprehensive Ala screen mutagenesis study in the editing domain of Aquifex aeolicus LeuRS (AaLeuRS) we found that the Y358A mutation reduced drastically the pretransfer editing but enhance the posttransfer editing. Based on the known structures we proposed a partial pretransfer editing model including the 3'-terminus of tRNA and Y358, following mutagenesis studies support our model and show that Y358 plays different but important roles in both pre- and posttransfer editing. On the other hand, we took advantage of the subtle difference of the key residue T273 of editing active site in recognition of pre- and posttransfer editing substrates, to produce mutations, typically T273R, which shut down the posttransfer editing without affecting pretransfer editing. In summary, we isolated the two tRNA-dependent editing pathways with the Y358A and T273R AaLeuRS mutants, respectively. And we proposed the possible conformation of 3'-terminus of tRNA in pretransfer editing, which was one of the most doubtful and interesting problems in editing.Mutations of R322 in the AaLeuRS editing domain interrupt the aminoacylation ability of the enzyme. Kinetics analysis indicated that the conformation of aminoacylation active site is affected by the mutations so that the binding and catalysis of the enzyme for ATP and amino acids are affected. The following assays proved that the occurring of editing in the editing domain will suppress the aminoacylation in the aminoacylation domain 30? far off, and the suppressive signal is conducted by R322. The deficiency in editing will enhance the suppression, indicating that the suppression of aminoacylation by editing might be a negative feedback mechanism to guarantee the fidelity of the aminoacylation. These results demonstrated the interdomain communication in LeuRS for the genetic fidelity.AatRNALeu has different binding manners with AaLeuRS as shown by our using enzymatic probe based footprinting study. The absolutely conserved A35 (the second base of anticodon) of AatRNALeu was protected by AaLeuRS in one case but not in the other. Unlike that in other tRNA systems, A35 of AatRNALeu was proved dispensable in aminoacylation reaction. However, the A35U tRNA mutant dramatically reduces the ability to trigger the total editing of AaLeuRS while enhances the posttransfer editing, indicating that A35 is an important recognition element by AaLeuRS in pretransfer editing. These results will improve the understanding of the mechanism of pretransfer editing and the co-evolution of tRNALeu/LeuRS.