Gel mobility shift experiments. (A) Binding reactions with HsPum (0, 0.0062, 0.0125, 0.025, 0.05, 0.10, 0.21, 0.41, 0.83, 1.65, and 3.3 μM) and RNAs bearing the indicated NRE1 sites. (B) Binding reactions with HsPum (0, 0.05, 0.10, 0.21, 0.41, 0.83, 1.65, and 3.3 μM) and RNAs bearing the indicated NRE2 sites. (C) Binding reactions with DmPum (0, 0.025, 0.05, 0.10, 0.21, 0.41, 0.83, 1.65, and 3.3 μM) and RNAs bearing the indicated NRE2 sites. Reactions with the Box B AUACUA mutant contained 0, 0.025, 0.08, 0.26, 0.52, 1.03 μM DmPum. In all three panels, the presumptive 1 Pum:1 NRE and 2 Pum:1 NRE complexes are indicated with arrowheads and asterisks, respectively.
