Structure and mechanism of a bacterial t(6)A biosynthesis system

The universal N(6)-threonylcarbamoyladenosine (t(6)A) modification at position 37 of ANN-decoding tRNAs is central to translational fidelity. In bacteria, t(6)A biosynthesis is catalyzed by the proteins TsaB, TsaC/TsaC2, TsaD and TsaE. Despite intense research, the molecular mechanisms underlying t(6)A biosynthesis are poorly understood. Here, we report biochemical and biophysical studies of the t(6)A biosynthesis system from Thermotoga maritima. Small angle X-ray scattering analysis reveals a symmetric 2:2 stoichiometric complex of TsaB and TsaD (TsaB(2)D(2)), as well as 2:2:2 complex (TsaB(2)D(2)E(2)), in which TsaB acts as a dimerization module, similar to the role of Pcc1 in the archaeal system. The TsaB(2)D(2) complex is the minimal platform for the binding of one tRNA molecule, which can then accommodate a single TsaE subunit. Kinetic data demonstrate that TsaB(2)D(2) alone, and a TsaB(2)D(2)E(1) complex with TsaE mutants deficient in adenosine triphosphatase (ATPase) activity, can catalyze only a single cycle of t(6)A synthesis, while gel shift experiments provide evidence that the role of TsaE-catalyzed ATP hydrolysis occurs after the release of product tRNA. Based on these results, we propose a model for t(6)A biosynthesis in bacteria.