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.

Cellular and Molecular Microbiology Campus La Plaine Building BC Room 1C4203 Boulevard du Triomphe 1050 Brussels Belgium

Cellular and Molecular Microbiology Campus La Plaine Building BC Room 1C4203 Boulevard du Triomphe 1050 Brussels Belgium; WELBIO Avenue Hippocrate 75 1200 Brussels Belgium

Department of Chemistry and Biotechnology Graduate School of Engineering University of Tokyo Bunkyo ku Tokyo 113 8656 Japan

Department of Experimental Medical Science Lund University 221 00 Lund Sweden

Department of Experimental Medical Science Lund University 221 00 Lund Sweden; University of Tartu Institute of Technology 50411 Tartu Estonia; Laboratory for Molecular Infection Medicine Sweden Umeå University 901 87 Umeå Sweden

Faculty of Life Sciences Kyoto Sangyo University Kamigamo Motoyama Kita ku Kyoto 603 8555 Japan

Institute of Organic Chemistry and Biochemistry Academy of Sciences of the Czech Republic v v i Flemingovonam 2 166 10 Prague 6 Czech Republic

Laboratory for Molecular Infection Medicine Sweden Umeå University 901 87 Umeå Sweden; Department of Clinical Microbiology Rigshospitalet 2200 Copenhagen Denmark

University of Tartu Institute of Technology 50411 Tartu Estonia

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