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 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 1^st, 2^nd, 4^th and 5^th base pairs the stem act as the specificity determinants. We show that the toxSASs PhRel2 and CapRel^SJ46 differ in tRNA specificity from FaRel2, and rationalise this through structural modelling: 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 defences, we hypothesise that highly evolvable modular tRNA recognition allows for the escape of viral countermeasures through tRNA substrate specificity switching.