Catalytic and regulatory basis of tRNA t⁶A modification by the KEOPS complex

N⁶-threonylcarbamoyladenosine (t⁶A) at tRNA position 37 is essential for translational fidelity and cellular homeostasis. The multi-subunit KEOPS complex catalyzes t⁶A formation in Archaea and Eukarya. Here we present cryo-EM structures of C. elegans KEOPS in its apo and tRNA-bound states. tRNA binding induces concerted conformational rearrangements, distorting the anticodon loop to project A37 into the Kae1 active site. Kae1 recognizes the conserved G10-C25 pair and 36-UAA-38 motif. Bud32 directly contacts the anticodon and acceptor arms, coupling ATP hydrolysis to t⁶A catalysis in the distant Kae1 active site through long-range conformational changes. Cgi121 enhances catalytic efficiency through cooperative binding with Bud32 and the tRNA 3' CCA. Pcc1 stabilizes the anticodon loop and mediates KEOPS dimerization, enhancing tRNA binding and t⁶A activity. GAMOS-associated mutations cluster at functional hotspots within the KEOPS-tRNA complex. This study provides a structural framework for understanding KEOPS mechanism and its cellular roles.