3 CONCLUSION
From an analysis of available crystal structures of bacterial GluRSs, including Eco -GluRS that we report here, and bacterial whole genomes to identify the presence or absence of GlnRS and gatCAB, we have identified a loop, spanning between Helix 8 and Helix 9, to be responsible for tRNAGln-discrimination by some but not all bacterial GluRSs. Specifically we show that the loop, which interacts with the D-helix of tRNAGlx, broadly adopts two conformations: [>>t] (towards RNA) or [t>>] (away from RNA). The [t>>] conformation is compatible with the two canonical conformations of the D-helix: augmented and non-augmented. The [>>t] conformation, which appears in various forms (Figure 7), displays a carboxylic side-chain towards tRNAGlx, that participates in favorable interactions with the augmented D-helix but either loses the favorable interactions or generates steric clashes (or both) when interacting with the non-augmented D Helix. The [>>t] conformation is most prevalent in proteobacterial GluRSs which also dominantly possess D-helix augmented tRNAGlu and non-augmented tRNAGln. That tRNAGln-discrimination by proteobacterial GluRSs are mediated by the [>>t] conformation was validated with known experimental data on tRNAGln-discrimination by GluRS or from genomic compulsions of a GluRS to be tRNAGln-discriminatory. Until now, the structural features of tRNAGln-discrimination by proteobacterial GluRSs were known to be present in tRNAGln, manifested as augmented versus non-augmented D-helix. This work, for the first time, identifies structural features on GluRS that complements the D-helix signatures on tRNAGln that gives rise to tRNAGln-discrimination by proteobacterial GluRSs.