RelA-SpoT Homolog (RSH) enzymes control bacterial physiology through synthesis and degradation of the nucleotide alarmone (p)ppGpp. We recently discovered multiple families of small alarmone synthetase (SAS) RSH acting as toxins of toxin-antitoxin (TA) modules, with the FaRel subfamily of toxSAS abrogating bacterial growth by producing an analog of (p)ppGpp, (pp)pApp. Here we probe the mechanism of growth arrest used by four experimentally unexplored subfamilies of toxSAS: FaRel2, PhRel, PhRel2, and CapRel. Surprisingly, all these toxins specifically inhibit protein synthesis. To do so, they transfer a pyrophosphate moiety from ATP to the tRNA 3′ CCA. The modification inhibits both tRNA aminoacylation and the sensing of cellular amino acid starvation by the ribosome-associated RSH RelA. Conversely, we show that some small alarmone hydrolase (SAH) RSH enzymes can reverse the pyrophosphorylation of tRNA to counter the growth inhibition by toxSAS. Collectively, we establish RSHs as RNA-modifying enzymes. [Display omitted] • Several families of toxSAS RSH toxins specifically inhibit protein synthesis • Translation-inhibiting toxSASs transfer pyrophosphate from ATP to the tRNA 3′ CCA • 3′ CCA pyrophosphorylation abrogates tRNA aminoacylation • Some SAH RSH enzymes can reverse the pyrophosphorylation of tRNA The RelA-SpoT Homolog (RSH) protein family contains both housekeeping stress-response enzymes and toxins of toxin-antitoxin systems (toxSAS). Kurata et al. uncover the mechanism of toxicity used by translation-inhibiting toxSAS. Unlike other RSHs synthetases that produce alarmone nucleotides, most toxSASs are RNA-modifying enzymes that pyrophosphate the tRNA 3′ CCA end. [ABSTRACT FROM AUTHOR]