(A) Electrophoretic mobility shift assays with 32P-labeled TAR and increasing concentrations of Tat-P-TEFb, or Tat-P-TEFb + AFF432–67, Tat-P-TEFb + AFF42–73, Tat-P-TEFb + AFF42–98. Control assays (bottom right) with P-TEFb and AFF42–73 showed no shifts for TAR. Half of TAR was shifted with 35–40 nM Tat-P-TEFb complex. In the presence of excess AFF4 fragments 32–67, 2–73, or 2–98, 50% of TAR was shifted by 1.1 nM Tat-AFF4-P-TEFb complex. (B) Calculated electrostatic surface potential of Tat-AFF4-CycT1 centered on the CycT1 TRM. The ribbon diagram (right) is in the same orientation as the surface representation (left). This orientation converts into the orientation in Figure 1A by consecutive rotations around y (70°) and z (−35°). CDK9 was omitted from the surface figure (left) to focus on the TAR interaction region. Solvent-exposed CycT1 residues K253, R254, N257, W258, R259, and Tat R49, which have no side-chain electron density, were modeled in the most common orientation. The electrostatic potential, calculated using APBS (Baker et al., 2001) was applied to color the solvent excluded surface of Tat-AFF4-CycT1 in Chimera (Pettersen et al., 2004) from −5 kbTe−1 (red) to +5 kbTe−1 (blue). CycT1 residues were labeled in black, Tat residues in red. The TRM region forms a positively charged patch on the SEC surface close to the disordered Tat ARM, which follows Tat R49.