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 this unique tRNA ^Glx-specificity is important. Mutational studies have hinted at hotspot residues, both on tRNA ^Glx and GluRS, that play crucial roles in tRNA ^Glx-specificity. But the underlying structural basis remains unexplored. 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), the proteobacterial biochemical data have often been interpreted in the context of non-proteobacterial GluRS structures. Marked differences between proteo- and non-proteobacterial GluRSs have been demonstrated and therefore it is important that tRNA ^Glx-specificity be understood vis-a-vis proteobacterial GluRS structures. Towards this goal we have solved the crystal structure of GluRS from E. coli. Using the solved structure and several other currently available proteo- and non-proteobacterial GluRS crystal structures, we have probed the structural basis of tRNA ^Glx-specificity of bacterial GluRSs. Specifically, our analysis suggests a unique role played by a tRNA ^Glx D-helix contacting loop of GluRS in modulation of tRNA ^Gln-specificity. While earlier studies had identified functional hotspots on tRNA ^Glx that controlled tRNA ^Glx-specificity of GluRS, this is the first report of complementary signatures of tRNA ^Glx-specificity in GluRS.