Fig. 6. Rrn3 interacts with Rrn6. (A) Two-hybrid interactions between Rrn3 and Rrn6. Strain Y190 was transformed with two plasmids, one allowing expression of Rrn3 fused to the Gal4 DBD, the other expression of Rrn6, Rrn6[772–894] or Rrn6[865–897] fused to the Gal4 AD. Activation of the lacZ and HIS3 reporter genes was monitored by staining the cells in the presence of XGal or replicating the cells on 3AT-containing medium, respectively. (B) The C-terminal domain of Rrn6 binds to immobilized Rrn3. Escherichia coli extracts containing (+) or not (–) recombinant HA-tagged Rrn3 were loaded onto protein G–Sepharose columns coated with 12CA5 anti-HA antibodies, then in vitro synthesized [³⁵S]Rrn6[C1] (Load) were applied to the columns. Proteins eluted by competition with purified HA-peptide were subjected to SDS–PAGE and analysed by autoradiography. (C) Full-length Rrn3 binds to the immobilized C-terminal domain of Rrn6. Escherichia coli extracts containing GST (–) or GST–Rrn6[772–894] fusion protein (+) were bound to glutathione–Sepharose columns. After extensive washing, partially purified recombinant HA-tagged Rrn3 (Load) was chromatographed onto the columns. Proteins eluted with glutathione were subjected to SDS–PAGE and the presence of HA-Rrn3 analysed by western blotting using monoclonal anti-HA antibodies.