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.