Toxic small alarmone synthetase FaRel2 inhibits translation by pyrophosphorylating tRNA ^Gly and tRNA ^Thr

Translation-targeting toxic small alarmone synthetases (toxSAS) are effectors of bacterial toxin-antitoxin systems that pyrophosphorylate the 3'-CCA end of transfer RNA (tRNA) to prevent aminoacylation. toxSAS are implicated in antiphage immunity: Phage detection triggers the toxSAS activity to shut down viral production. We show that the toxSAS FaRel2 inspects the tRNA acceptor stem to specifically select tRNA^Gly and tRNA^Thr. The first, second, fourth, and fifth base pairs of the stem act as the specificity determinants. We show that the toxSASs PhRel2 and CapRel^SJ46 differ in tRNA specificity from FaRel2 and rationalize this through structural modeling: While the universal 3'-CCA end slots into a highly conserved CCA recognition groove, the acceptor stem recognition region is variable across toxSAS diversity. As phages use tRNA isoacceptors to overcome tRNA-targeting defenses, we hypothesize that highly evolvable modular tRNA recognition allows for the escape of viral countermeasures through tRNA substrate specificity switching.