Adaptation of a eukaryote-like ProRS to a prokaryote-like tRNA^Pro

Prolyl-tRNA synthetases (ProRSs) are unique among aminoacyl-tRNA synthetases (aaRSs) in having two distinct structural architectures across different organisms: prokaryote-like (P-type) and eukaryote/archaeon-like (E-type). Interestingly, Bacillus thuringiensis harbors both types, with P-type (BtProRS1) and E-type ProRS (BtProRS2) coexisting. Despite their differences, both enzymes are constitutively expressed and functional in vivo. Similar to BtProRS1, BtProRS2 selectively charges the P-type tRNA^Pro and displays higher halofuginone tolerance than canonical E-type ProRS. However, these two isozymes recognize the primary identity elements of the P-type tRNA^Pro—G72 and A73 in the acceptor stem—through distinct mechanisms. Moreover, BtProRS2 exhibits significantly higher tolerance to stresses (such as heat, hydrogen peroxide, and dithiothreitol) than BtProRS1 does. This study underscores how an E-type ProRS adapts to a P-type tRNA^Pro and how it may contribute to the bacterium’s survival under stress conditions.