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

Abstract

The universal N⁶-threonylcarbamoyladenosine (t⁶A) at position 37 of tRNAs is one of core post-transcriptional modifications that are needed for promoting translational fidelity. In bacteria, TsaC utilizes 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 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 recombinant TsaC, TsaD-TsaB and TsaE from thermophilic Aquifex aeolicus and determined crystal structures of apo-form and ADP-bound form of TsaD₂B₂ tetramer. Our TsaD₂B₂-TsaE-tRNA model coupled 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 TsaD₂B₂ or TsaDB possesses a basal divalent ion-dependent t⁶A-catalytic activity that is stimulated by TsaE at the cost of ATP consumption. Our data suggest that 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 release of tRNA t⁶A and AMP. ATP hydrolysis-driven dissociation of TsaE from TsaDB resets an active conformation of TsaDB. Dimerization of thermophilic TsaDB enhances thermostability and promotes t⁶A-catalytic activity of TsaD₂B₂-tRNA, of which GC base pairs in anticodon stem are needed for correct folding of thermophilic tRNA at higher temperatures.