Reversible RNA phosphorylation stabilizes tRNA for cellular thermotolerance.

Post-transcriptional modifications have critical roles in tRNA stability and function. In thermophiles, tRNAs are heavily modified to maintain their thermal stability under extreme growth temperatures. Here we identified 2'-phosphouridine (U) at position 47 of tRNAs from thermophilic archaea. U47 confers thermal stability and nuclease resistance to tRNAs. Atomic structures of native archaeal tRNA showed a unique metastable core structure stabilized by U47. The 2'-phosphate of U47 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 U47 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 U47 in vitro and in vivo. Taken together, our findings show that U47 is a reversible RNA modification mediated by ArkI and KptA that fine-tunes the structural rigidity of tRNAs under extreme environmental conditions.