Signatures of tRNAGlx‐specificity in proteobacterial glutamyl‐tRNA synthetases

The canonical function of glutamyl‐tRNA synthetase (GluRS) is to glutamylate tRNAGlu. Yet not all bacterial GluRSs glutamylate tRNAGlu; many glutamylate both tRNAGlu and tRNAGln, while some glutamylate only tRNAGln and not the cognate substrate tRNAGlu. Understanding the basis of the unique specificity of tRNAGlx is important. Mutational studies have hinted at hotspot residues, both on tRNAGlx and GluRS, which play crucial roles in tRNAGlx‐specificity. However, its underlying structural basis remains unexplored. The majority of biochemical studies related to tRNAGlx‐specificity have been performed on GluRS from Escherichia coli and other proteobacterial species. However, since the early crystal structures of GluRS and tRNAGlu‐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 tRNAGlx‐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 tRNAGlx‐specificity of bacterial GluRSs. Specifically, our analyses suggest a unique role played by the tRNAGlx D‐helix contacting loop of GluRS in the modulation of tRNAGln‐specificity. While earlier studies have identified functional hotspots on tRNAGlx that control the tRNAGlx‐specificity of GluRS, this is the first report of complementary signatures of tRNAGlx‐specificity in GluRS.