A minimalist mitochondrial threonyl-tRNA synthetase exhibits tRNA-isoacceptor specificity during proofreading

Abstract

Yeast mitochondria contain a minimalist threonyltRNA synthetase (ThrRS) composed only of the catalytic core and tRNA binding domain but lacking the entire editing domain. Besides the usual tRNA Thr 2, some budding yeasts, such as Saccharomyces cerevisiae, also contain a non-canonical tRNA Thr 1 with an enlarged 8-nucleotide anticodon loop, reprograming the usual leucine CUN codons to threonine. This raises interesting questions about the aminoacylation fidelity of such ThrRSs and the possible contribution of the two tRNA Thr s during editing. Here, we found that, despite the absence of the editing domain, S. cerevisiae mitochondrial ThrRS (ScmtThrRS) harbors a tRNA-dependent pretransfer editing activity. Remarkably, only the usual tRNA Thr 2 stimulated pre-transfer editing, thus, establishing the first example of a synthetase exhibiting tRNA-isoacceptor specificity during pre-transfer editing. We also showed that the failure of tRNA Thr 1 to stimulate tRNA-dependent pre-transfer editing was due to the lack of an editing domain. Using assays of the complementation of aScmtThrRS gene knockout strain, we showed that the catalytic core and tRNA binding domain of ScmtThrRS co-evolved to recognize the unusual tRNA Thr 1. In combination, the results provide insights into the tRNA-dependent editing process and suggest that tRNA-dependent pretransfer editing takes place in the aminoacylation catalytic core.