Reversible RNA phosphorylation stabilizes tRNA for cellular thermotolerance

Post-transcriptional modifications have critical roles in tRNA stability and function^1-4. In thermophiles, tRNAs are heavily modified to maintain their thermal stability under extreme growth temperatures^5,6. Here we identified 2'-phosphouridine (U^p) at position 47 of tRNAs from thermophilic archaea. U^p47 confers thermal stability and nuclease resistance to tRNAs. Atomic structures of native archaeal tRNA showed a unique metastable core structure stabilized by U^p47. The 2'-phosphate of U^p47 protrudes from the tRNA core and prevents backbone rotation during thermal denaturation. In addition, we identified the arkI gene, which encodes an archaeal RNA kinase responsible for U^p47 formation. Structural studies showed that ArkI has a non-canonical kinase motif surrounded by a positively charged patch for tRNA binding. A knockout strain of arkI grew slowly at high temperatures and exhibited a synthetic growth defect when a second tRNA-modifying enzyme was depleted. We also identified an archaeal homologue of KptA as an eraser that efficiently dephosphorylates U^p47 in vitro and in vivo. Taken together, our findings show that U^p47 is a reversible RNA modification mediated by ArkI and KptA that fine-tunes the structural rigidity of tRNAs under extreme environmental conditions.