Anticodon loop remodeling and D-stem shape drive the specific recognition of ANN-decoding tRNAs for t6A modification.

N6-threonylcarbamoyladenosine (t6A) is a universal transfer RNA (tRNA) modification essential for translational fidelity. The modification is installed at position 37 of ANN-decoding tRNAs (N is A, U, G, or C) by transfer of a threonylcarbamoyl moiety from a pathway intermediate, catalyzed in bacteria by the TsaBD complex. Despite the strict requirement for the 36-UAA-38 sequence in substrate tRNAs, the structural basis for this specificity has remained unclear. We determined cryo-EM structures of the Thermotoga maritima t6A synthase bound to unmodified and to natively modified tRNA carrying the t6A37 modification, at a nominal resolution of 3.2 Å. In both structures, A37 is positioned in the active site, and the anticodon loop is remodeled into a zig-zag conformation not previously observed in tRNA, stabilized by conserved interactions with the RNA backbone at the TsaD/TsaB interface. The wobble and middle anticodon bases occupy shallow surface pockets without base-specific contacts, explaining tolerance to base identity at these positions. In contrast, U36 and A38 are recognized indirectly through formation of a base triple with U32. Additional D-stem contacts provide a second mode of indirect readout. Together with mutagenesis data, the structures reveal the molecular basis for restriction of t6A37 to ANN-decoding tRNAs and the requirement for A38.