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.
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