Equilibrium- and competition-binding assays for wild-type and mutant LtrA proteins with N- or C-terminal truncations. (A) Equilibrium-binding assays. 32P-labeled DIVa-1 RNA (5 pM) was incubated with increasing concentrations of wild-type and the indicated mutant LtrA proteins in 450 NMT medium for 60 min at 30°C (see Materials and Methods). The mixture was then filtered through layered nitrocellulose and Hybond-N nylon membranes, and radioactive RNA bound to the filters was quantified by using a PhosphorImager. The percentage of input RNA bound was plotted as a function of protein concentrations, and the plots were fit by a hyperbola to obtain Kd values. Apparent Kds ± the standard error of the fit for the curves shown are indicated. Similar results were obtained in three repeats of the experiment. A fit by the Hill equation gave n = 1, indicating that binding of LtrA to DIVa-1 RNA is not cooperative (data not shown). (B) Competition-binding assays. A mixture containing 100 nM each of 32P-labeled DIVa-2 RNA (135 nt; control [C]) and wild-type or mutant DIVa-1 RNA (119 nt; experimental [E]) was incubated with limiting amounts of wild-type or the indicated mutant LtrA proteins (20 nM) in 450 NMT for 60 min at 30°C. After filtering through nitrocellulose, the bound RNAs were recovered by phenol-CIA extraction and analyzed by electrophoresis in a denaturing 4% polyacrylamide gel, which was dried and quantified with a PhosphorImager. Input shows individual RNAs or the mixture processed in the same way but without the filtration step. The control (C) RNA is wild type; the experimental (E) RNA is indicated above each lane; and the protein is indicated below the lanes. The binding ratios calculated as described in the Materials and Methods are indicated below the lanes for competition assays.
