Figure 4.

RRE IIB binding by ZF2-Rev in vitro. (A) Gel shift assays of ZF2-Rev and Rev14_ala peptides with wild-type RRE IIB RNA and a C46-G74 mutant (that reverses a base pair critical for recognition; refs. 21, 26, and 27) at the peptide concentrations (nM) indicated. ZF2-Rev binds with a similar affinity and specificity as the helical Rev peptide; apparent binding constants are substantially tighter in the absence of competitor tRNA (21, 33). (B, Left) Zinc-dependent RRE IIB RNA binding: ZF2-Rev in the presence of 25 μM ZnCl₂ (■), in the absence of zinc and with addition of 0.5 mM EDTA (□), and ZF2-Rev (C5,8S) in the presence of 25 μM ZnCl₂ (●). (Right) Binding curves quantitated by PhosphorImager analyses and apparent dissociation constants (nM) estimated by fitting to standard binding equations.