FIG. 6.

Three-membrane sandwich experiments to demonstrate cooperative RNA binding by a truncated recombinant p33. The purified recombinant C10 protein (Fig. 7; the applied amount is shown on the left side) was incubated with ³²P-labeled region III(-) RNA probe for 30 min at 25°C, followed by filtering the reaction products through a three-membrane sandwich (36) in a slot blot apparatus. The membranes were then washed twice with (300 μl) binding buffer, air dried, and analyzed using a phosphorimager. The membranes were used in the following order: polysulfone on the top (column 1), nitrocellulose in the middle (column 2), and Hybond N+ on the bottom (column 3). Note that the unbound label at the highest protein concentration likely represents labeled ribonucleotides (which, in spite of a purification step, are still present in the RNA probe as a carryover from the labeling reaction) that were unbound were to protein C10.