Signatures of tRNA Glx -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.