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(p)47 confers thermal stability and nuclease resistance to tRNAs. Atomic structures of native archaeal tRNA showed a unique metastable core structure stabilized by U(p)47. The 2′-phosphate of U(p)47 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(p)47 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(p)47 in vitro and in vivo. Taken together, our findings show that U(p)47 is a reversible RNA modification mediated by ArkI and KptA that fine-tunes the structural rigidity of tRNAs under extreme environmental conditions.