(A) Rev dimer structure (Daugherty et al., 2010b) with the higher-order oligomerization disrupting mutations, L12S and L60R shown in red. Also shown is the surface-entropy reducing mutation, E47A in blue or green. (B) RRE IIB40 is a derivative of the stem II three-helix junction and contains two adjacent Rev-binding sites (red). (C) Gel-shift assays comparing binding of 32P-labeled RRE-stem II or IIB40 to Rev. Free, F, monomer, M and dimer, D complexes are indicated. A doublet/smeared band is observed for the monomer complex with both RNAs and is indicative of conformational heterogeneity. (D) Binding curves calculated from gel-shift assays in (C). Apparent dissociation constants, Kd and Hill coefficient (n) were determined using the equation: Fraction of RNA bound = [Rev]n/(Kdn + [Rev]n) as mean ± s.d. of two replicates.