Trans-interactions between TRAF6 and donor-Ub are critical for Ub transfer. (A) Superimposed gel filtration profiles of TRAF6 wild-type and mutants, showing the preservation of dimeric states in the mutants. The TRAF6 constructs, containing RING and ZF1-3, have a theoretical dimer molecular mass of 37 kDa. mAU, milli absorption units. (B) Promotion of polyubiquitin chain synthesis by wild-type and mutant TRAF6 in the presence of E1, the E2 complex Ubc13/Uev1A, and ubiquitin. (C) Promotion of polyubiquitin chain synthesis by wild-type and mutant TRAF6 at different time points.