Conformational communication mediates the reset step in t(6)A biosynthesis

The universally conserved N(6)-threonylcarbamoyladenosine (t(6)A) modification of tRNA is essential for translational fidelity. In bacteria, t(6)A biosynthesis starts with the TsaC/TsaC2-catalyzed synthesis of the intermediate threonylcarbamoyl adenylate (TC–AMP), followed by transfer of the threonylcarbamoyl (TC) moiety to adenine-37 of tRNA by the TC-transfer complex comprised of TsaB, TsaD and TsaE subunits and possessing an ATPase activity required for multi-turnover of the t(6)A cycle. We report a 2.5-Å crystal structure of the T. maritima TC-transfer complex (TmTsaB(2)D(2)E(2)) bound to Mg(2+)-ATP in the ATPase site, and substrate analog carboxy-AMP in the TC-transfer site. Site directed mutagenesis results show that residues in the conserved Switch I and Switch II motifs of TsaE mediate the ATP hydrolysis-driven reactivation/reset step of the t(6)A cycle. Further, SAXS analysis of the TmTsaB(2)D(2)-tRNA complex in solution reveals bound tRNA lodged in the TsaE binding cavity, confirming our previous biochemical data. Based on the crystal structure and molecular docking of TC–AMP and adenine-37 in the TC-transfer site, we propose a model for the mechanism of TC transfer by this universal biosynthetic system.