Structure–function analysis of tRNA t6A-catalysis, assembly, and thermostability of Aquifex aeolicus TsaD2B2 tetramer in complex with TsaE

The universal N⁶-threonylcarbamoyladenosine (t⁶A) at position 37 of tRNAs is one of the core post-transcriptional modifications that are needed for promoting translational fidelity. In bacteria, TsaC uses L-threonine, bicarbonate, and ATP to generate an intermediate threonylcarbamoyladenylate (TC-AMP), of which the TC moiety is transferred to N6 atom of tRNA A37 to generate t⁶A by TsaD with the support of TsaB and TsaE. TsaD and TsaB form a TsaDB dimer to which tRNA and TsaE are competitively bound. The catalytic mechanism of TsaD and auxiliary roles of TsaB and TsaE remain to be fully elucidated. In this study, we reconstituted tRNA t⁶A biosynthesis using TsaC, TsaD, TsaB, and TsaE from Aquifex aeolicus and determined crystal structures of apo-form and ADP-bound form of TsaD₂B₂ tetramer. Our TsaD₂B₂-TsaE-tRNA model coupled with functional validations reveal that the binding of tRNA or TsaE to TsaDB is regulated by C-terminal tail of TsaB and a helical hairpin α1-α2 of TsaD. A. aeolicus TsaDB possesses a basal t⁶A catalytic activity that is stimulated by TsaE at the cost of ATP consumption. Our data suggest that the binding of TsaE to TsaDB induces conformational changes of α1, α2, α6, α7, and α8 of TsaD and C-terminal tail of TsaB, leading to the release of tRNA t⁶A and AMP. ATP-mediated binding of TsaE to TsaDB resets a t⁶A active conformation of TsaDB. Dimerization of TsaDB enhances thermostability and promotes t⁶A catalysis of TsaD₂B₂-tRNA, of which GC base pairs in anticodon stem are needed for the correct folding of thermophilic tRNA at higher temperatures.