Binding synergy as an essential step for tRNA editing and modification enzyme codependence in Trypanosoma brucei

TbADAT2/3 catalytic residues do not contribute to increased affinity of TbTrm140 for tRNA. (A) EMSA of TbTrm140 to assess binding to tRNA^Thr_CGU in the presence of two TbADAT2/3 catalytic mutants (E92A) and (B) (C291A). (C) EMSA of TbTrm140 to tRNA^Thr_CGU in the presence of TbADAT2/3 C-terminal deletion binding mutant. In each panel, lanes 1 and 2 show a no-enzyme control reaction and a control reaction with no mutant TbADAT2/3 added, respectively. Lanes 3–7 show an increasing concentration of TbTrm140 (0.04, 0.08, 0.16, 0.32, and 0.56 µM, respectively). The bottom panels show the single-ligand binding isotherms used to calculate the individual K_dapp. Each graph represents at least five independent replicates.