Divergent anticodon recognition in contrasting glutamyl-tRNA synthetases

The aminoacyl-tRNA synthetases (AARSs) lie at the heart of protein translation. Each of these enzymes catalyzes the attachment of a single cognate amino acid to one or more cognate transfer RNA (tRNAs) with high fidelity - these aminoacyl-tRNAs are subsequently delivered to the ribosome for protein biosynthesis. One of the hallmarks of the AARSs is their ability to discriminate between different tRNAs, rejecting non-cognate tRNAs in nearly all cases.1 In general, one AARS corresponds to each of the twenty encoded amino acids.1 For example, a canonical, discriminating glutamyl-tRNA synthetase (D-GluRS) catalyzes the formation of Glu-tRNAGlu (Eq. 1). Recent efforts have elucidated a few key exceptions to this general rule, via the identification of organisms that generate a complete set of aminoacyl-tRNAs with an incomplete set of AARSs.2, 3 In fact, glutaminyl-tRNA synthetase (GlnRS) is missing from all archaea and most bacteria, organelles and chloroplasts evaluated to date.1 In these organisms, the specificity of GluRS is relaxed or non-discriminatory (ND-GluRS). ND-GluRSs are essential for the biosynthesis of their cognate substrate Glu-tRNAGlu (Eq. 1) and for the misacylation of the non-cognate tRNAGln, to generate Glu-tRNAGln (Eq. 2). Glu-tRNAGln is subsequently transamidatively converted to Gln-tRNAGln by a Glu-tRNAGln glutamine-dependent amidotransferase, Glu-Adt (Eq. 3).4-7 Glu+ATP+tRNAGlu+GluRS→AMP+PPi+Glu−tRNAGlu Eq. 1 Glu+ATP+tRNAGln+ND−GluRS→AMP+PPi+Glu−tRNAGln Eq. 2 Glu−tRNAGln+ATP+Gln+Glu−Adt→Glu+ADP+Pi+Gln−tRNAGln Eq. 3 The recent discovery and characterization of pairs of co-existing GluRSs in a few bacteria necessitated the expansion of GluRS types to include GluRS1 and GluRS2.8, 9 In Helicobacter pylori, GluRS2 is unexpectedly non-canonical as its sole catalytic role is to biosynthesize Glu-tRNAGln and it has lost the ability to aminoacylate its “cognate” tRNAGlu. In contrast, Hp GluRS1 is a canonical D-GluRS that aminoacylates the two tRNAGlu isoacceptors of H. pylori (tRNAGlu1 and tRNAGlu2).8, 9 Interestingly, the Acidithiobacillus ferrooxidans (Af) GluRS2 is specific only for tRNAGlnUUG, while Af GluRS1 aminoacylates both tRNAGlu isoacceptors and the remaining tRNAGln isoacceptor, tRNAGlnCUG.9 Soll and coworkers have also demonstrated a correlation between the size of the D-stem helix in tRNAGlu and tRNAGln isoacceptors and the complementary tRNA specificities of GluRS1 and GluRS2 in both H. pylori and in A. ferrooxidans.9 Genes encoding for co-existing GluRS1/GluRS2 pairs have been identified in more than 30 bacterial genomes. We have proposed that GluRS2 could represent a mid-point in an attempt by bacteria to evolve a bacterial GlnRS, unique from known GlnRSs8 (which are all eukaryotic in origin10, 11). This proposal was based on the well-documented ancestral connection between eukaryotic GluRS and GlnRS paralogs10-12 and the fact that GluRS1 and GluRS2 are related by an ancestral gene duplication event.8, 9 Whether or not GluRS2 represents the precursor to a future bacterial GlnRS, the co-existence of pairs of closely related GluRSs offers an unprecedented opportunity to directly probe how subtle divergence in primary sequence can lead to the emergence of new tRNA specificities in evolution. Because species-dependent variations in tRNA sequences are irrelevant in these cases, insight can be gained into the subtle differences between GluRS1 and GluRS2 in a more straightforward manner than when comparing two GluRSs (e.g. a D-GluRS and an ND-GluRS) from different organisms. In this respect, we are broadly interested in understanding how H. pylori GluRS1 and GluRS2 specifically select only tRNAGlu or tRNAGln, respectively, with the particular goal of using these enzymes to explore specification within the genetic code. In this work, we report evidence for divergent evolution in the anticodon-binding domains (ABD) of GluRS1 versus GluRS2.