Co-occupancy of two Pumilio molecules on a single hunchback NRE

Fluorescence anisotropy. (A) Binding curves for human Pum Puf domain (HsPum) with 5′-fluoroscein (5′-Fl)-labeled NRE1_wt, NRE2_wt, NRE1_MutA, NRE1_MutB, and NRE1_MutA+B RNAs. (B) Binding curves for Drosophila Pum Puf domain (DmPum) with 5′-Fl NRE1_wt, NRE1_MutA, and NRE1_MutB. On NRE1_MutA+B, the data were too noisy to be fit with an isotherm, probably because DmPum has a tendency to aggregate at the high concentrations required for the weak binding to NRE1_MutA+B. The experiments were performed as described in Materials and Methods. The errors were determined from the fit of the isotherms to the binding equations.