Aminoacyl-tRNA synthetases: Selection of expression approaches and functional analysis of the N-terminal extension

Aminoacyl-tRNA synthetases catalyze the first step in protein biosynthesis and play a key role in translation of the genetic code. In comparison to their prokaryotic counterparts, mammalian aminoacyl-tRNA synthetases associate as multi-synthetase complexes and contain highly charged amino-terminal extensions. The functions of the amino-terminal extensions have been the focus of intense investigation given that the extensions are dispensable for aminoacylation. Within the full-length protein or as an isolated domain, the three-dimensional structure of the amino-terminal extensions in mammalian aminoacyl-tRNA synthetases has not been determined. Mammalian enzymes such as human aspartyl-tRNA synthetase (hDRS) are frequently produced as inactive proteins in E. coli. A systematic comparison of the expression and purification of hDRS should provide useful guidance for selection of expression approaches. We expressed in E. coli ubiquitin- and SUMO-hDRS fusion proteins with biotin- or 6xHis-tags. Tagging of hDRS with ubiquitin or SUMO was necessary to express properly folded and biologically active enzyme. Ubiquitin, SUMO, biotin, and hexahistidine tags did not appreciably interfere with hDRS activity.; Functional analysis of the amino-terminal extension in hKRS was carried out using site-directed mutagenesis. Deletion of the amino-terminal extension in hKRS yielded hKRSDelta60 with reduced but comparable lysylation activity. Compared to full-length hKRS, elongation factor 1alpha (EFIalpha) bound to and enhanced the activity of hKRSDelta60 to a greater extent. Addition of p38, the scaffold protein of the multisynthetase complex, had no effect on the activity of either full-length hKRS or hKRSDelta60. Aminoacylation activity of full-length hKRS was more significantly enhanced compared to hKRSDelta60 by the presence of hDRS. These results report the first known activity enhancement of a mammalian aminoacyl-tRNA synthetase by another synthetase. Charged residues in the amino-terminal extension of hKRS that play a role in stability, tRNA binding, lysylation, and EF1alpha interaction were identified. Results indicate acidic residues in the amino-terminal extension in hKRS contributed to tRNA interaction consistent with the magnesium ion mediated RNA-protein interaction hypothesis. Basic residues participated in RNA-protein and protein-protein interactions. These studies provide insight into the structure and multiple functions of hKRS with specific regard to the newly evolved amino-terminal extension.