Signatures of tRNAGlx-specificity in proteobacterial glutamyl-tRNA synthetases.

The canonical function of glutamyl-tRNA synthetase (GluRS) is to glutamylate tRNA^Glu. Yet not all bacterial GluRSs glutamylate tRNA^Glu; many glutamylate both tRNA^Glu and tRNA^Gln, while some glutamylate only tRNA^Gln and not the cognate substrate tRNA^Glu. Understanding the basis of the unique specificity of tRNA^Glx is important. Mutational studies have hinted at hotspot residues, both on tRNA^Glx and GluRS, which play crucial roles in tRNA^Glx-specificity. However, its underlying structural basis remains unexplored. The majority of biochemical studies related to tRNA^Glx-specificity have been performed on GluRS from Escherichia coli and other proteobacterial species. However, since the early crystal structures of GluRS and tRNA^Glu-bound GluRS were from non-proteobacterial species (Thermus thermophilus), proteobacterial biochemical data have often been interpreted in the context of non-proteobacterial GluRS structures. Marked differences between proteobacterial and non-proteobacterial GluRSs have been demonstrated; therefore, it is important to understand tRNA^Glx-specificity vis-a-vis proteobacterial GluRS structures. To this end, we solved the crystal structure of a double mutant GluRS from E. coli. Using the solved structure and several other currently available proteo- and non-proteobacterial GluRS crystal structures, we probed the structural basis of the tRNA^Glx-specificity of bacterial GluRSs. Specifically, our analyses suggest a unique role played by the tRNA^Glx D-helix contacting loop of GluRS in the modulation of tRNA^Gln-specificity. While earlier studies have identified functional hotspots on tRNA^Glx that control the tRNA^Glx-specificity of GluRS, this is the first report of complementary signatures of tRNA^Glx-specificity in GluRS.