Figure 4

Qk-1-STAR binding to TGE RNA and consensus site mutants by fluorescence-polarization and EMSA. A. Plot showing polarization as a function of protein concentration for the direct titration of Qk-1 STAR Domain into fluorescein-labeled TGE RNA or the C21A TGE variant. B. Comparative plot of Qk-1-STAR titrations into radiolabeled TGE RNA, C21A TGE RNA or the 5' mutant TGE 28-mer. The gel mobility shift of Qk-1-STAR into C21A TGE RNA is shown with the bound and free species and the concentration of Qk-1-STAR through the titration indicated. C. Competition binding with the normalized polarization plotted against the concentration of unlabeled competitor 12-mer RNA. Shown are traces for the A21 12-mer half-site RNA and two mutant 12-mer sequences where each was titrated into a constant concentration of Qk-1-STAR in complex with C21A TGE RNA. D. Relative IC₅₀ values for each of the 12-mer mutants compared to the A21 half-site RNA.