PCA of H8-loop-H9 motifs in non-proteobacterial GluRS
The non-proteobacterial H8-L -H9 cluster of Figure 3A (cluster C) was analyzed further using a separate PCA. As can be seen from Figure 3C, the H8-L -H9 motifs, showed a bias in their appearance on the PC1-PC2 plane depending upon whether they belonged to tRNAGln-discriminating (red circles) or non-discriminating (blue circles) GluRS. However, there was substantial overlap. Since GluRS sequence variation can be highly phylum-specific, we then looked at only those cases where both discriminating and non-discriminating GluRSs were present within the same class. Two such classes, firmicutes (Fi ) and hyperthermophilic bacteria (Ht ) in our database contained GluRS of both kinds (T1-GluRS/T2-GluRS for Ht and D(+)-GluRS/ND-GluRS for Fi ). The GluRS H8-L -H9 motifs in these two classes showed a clear sequence difference depending on their tRNAGln-specificity. For Ht (Figure 3D) all five pairs of T1-GluRS1/T2-GluRS separated into two distinct clusters. Sequence comparison of the two groups (Figure S3A) showed the unique presence of HPE(D)GK sequence at the center of the H8-L -H9 loop in T2-GluRSs absent in T1-GluRSs. Similarly, for Fi (Figure 3E), all H8-L -H9 motifs were separated into two clusters depending upon the tRNAGln-specificity of the corresponding GluRSs. Sequence comparison of the two groups (Figure S3B) showed the unique presence of Gly in ND-GluRSs (FMA is an exception); the D-GluRSs either lacked the Gly residue (replaced by Thr, Glu, Asp or Asn) or contained a 2-residue (WR) insertion at the center of H8-L -H9 motif.