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.

• MeSH
• bakteriální toxiny genetika   metabolismus   farmakologie   MeSH
• fosforylace účinky léků   MeSH
• grampozitivní nesporulující tyčinky chemie   metabolismus   MeSH
• guanosinpentafosfát chemie   metabolismus   MeSH
• inhibitory syntézy proteinů farmakologie   MeSH
• ligasy chemie   genetika   metabolismus   MeSH
• proteosyntéza účinky léků   fyziologie   MeSH
• pyrofosfatasy MeSH
• ribozomy metabolismus   MeSH
• RNA transferová metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Under stressful conditions, bacterial RelA-SpoT Homolog (RSH) enzymes synthesize the alarmone (p)ppGpp, a nucleotide second messenger. (p)ppGpp rewires bacterial transcription and metabolism to cope with stress, and, at high concentrations, inhibits the process of protein synthesis and bacterial growth to save and redirect resources until conditions improve. Single-domain small alarmone synthetases (SASs) are RSH family members that contain the (p)ppGpp synthesis (SYNTH) domain, but lack the hydrolysis (HD) domain and regulatory C-terminal domains of the long RSHs such as Rel, RelA, and SpoT. We asked whether analysis of the genomic context of SASs can indicate possible functional roles. Indeed, multiple SAS subfamilies are encoded in widespread conserved bicistronic operon architectures that are reminiscent of those typically seen in toxin-antitoxin (TA) operons. We have validated five of these SASs as being toxic (toxSASs), with neutralization by the protein products of six neighboring antitoxin genes. The toxicity of Cellulomonas marina toxSAS FaRel is mediated by the accumulation of alarmones ppGpp and ppApp, and an associated depletion of cellular guanosine triphosphate and adenosine triphosphate pools, and is counteracted by its HD domain-containing antitoxin. Thus, the ToxSAS-antiToxSAS system with its multiple different antitoxins exemplifies how ancient nucleotide-based signaling mechanisms can be repurposed as TA modules during evolution, potentially multiple times independently.

• MeSH
• adeninnukleotidy metabolismus   MeSH
• Bacteria růst a vývoj   metabolismus   MeSH
• bakteriální proteiny metabolismus   MeSH
• databáze genetické MeSH
• fyziologický stres fyziologie   MeSH
• guanosinpentafosfát metabolismus   MeSH
• guanosintetrafosfát metabolismus   MeSH
• guanosintrifosfát metabolismus   MeSH
• ligasy metabolismus   MeSH
• pyrofosfatasy metabolismus   MeSH
• regulace genové exprese u bakterií genetika   MeSH
• signální transdukce MeSH
• systémy toxin-antitoxin fyziologie   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH