The universal N-threonylcarbamoyladenosine (tA) 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 tA 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 tA biosynthesis using TsaC, TsaD, TsaB, and TsaE from Aquifex aeolicus and determined crystal structures of apo-form and ADP-bound form of TsaDB tetramer. Our TsaDB-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 tA 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 tA and AMP. ATP-mediated binding of TsaE to TsaDB resets a tA active conformation of TsaDB. Dimerization of TsaDB enhances thermostability and promotes tA catalysis of TsaDB-tRNA, of which GC base pairs in anticodon stem are needed for the correct folding of thermophilic tRNA at higher temperatures.