dc.description.abstract | Prolyl-tRNA synthetase (ProRS) is an enzyme that activates proline and attaches it to tRNAPro for protein synthesis. ProRS belongs to class IIa aminoacyl-tRNA synthetase, containing three conserved sequence domains (motifs 1-3). Based on functional domains, ProRS itself is divided into two types: eukaryotic/archaea-like type (E-type), which is characterized by the presence of a C-terminal extension domain (CTD), and prokaryotic-like type (P-type), which is characterized by the presence of an insertion domain (INS) between motifs 2 and 3. E-type ProRSs are primarily found in archaea, eukaryotes (cytoplasm and plant organelle), and some bacteria, while P-type ProRSs are found in most bacteria and mitochondria of eukaryotes. Phylogenetic analysis of ProRS showed that almost all bacteria contain one P-type ProRS and one P-type tRNAPro. However, the bacterium Bacillus thuringiensis contains both types of ProRS, a P-type (BtProRS1) and an E-type ProRS (BtProRS2), and a P-type tRNAPro. BtProRS1 contains an INS but lacks a CTD. On the contrary, BtProRS2 contains a CTD but lacks an INS. To investigate the tRNA specificities of these two ProRS enzymes, a heterologous complementation assay using a yeast ProS2 knockout strain and an aminoacylation assay using purified BtProRSs and tRNAsPro as substrates were carried out. We demonstrated herein that both BtProRS1 and BtProRS2 could rescue the yeast mitochondrial ProRS KO strain, suggesting that both enzymes can charge the P-type yeast mitochondrial tRNAPro to a level sufficient to maintain normal mitochondrial function. Unfortunately, neither BtProRS1 nor BtProRS2 charged yeast total tRNA, E. coli total tRNA, or BttRNAPro. Further phylogenetic analysis revealed that not all Bacillus species contain both types of ProRS. The biological significance of this finding will be further discussed. | en_US |