FIGURE 4.

Identification of RNA species that are required for the SP120-topo IIβ interaction. A, in vitro binding assay was performed as in Fig. 3C, except that endogenous RNA bound to SP120 was digested with RNase and replaced by the purified RNA preparations as indicated. FLAG-topo IIβ was detected by Western blotting with anti-FLAG antibody, whereas Myc-SP120 recombinants (WT and C187) were visualized directly by SYPRO Ruby staining. Asterisk indicates the antibody's light chain. B, RNA added to the binding reaction (input) and the RNA recovered from the protein complex were separated by 1% agarose gel electrophoresis under denatured conditions and detected by staining with SYBR Green II. Asterisks indicate rRNAs. C, binding experiment was performed as in A with 1 μg of synthetic polyribonucleotides instead of natural RNAs. Proteins on beads were detected by Western blotting with anti-tag antibodies. The asterisk indicates the light chain of the IP antibody.