Fig. 4. Binding of Rex to cydP1. (A) DNase I footprinting of the Rex–cydP1 complex. The 360 bp cydP1 fragment was 5′ end-labelled on the bottom (template) strand and mixed with increasing concentrations of Rex, prior to DNase I treatment, as follows: lane 3, no added Rex; lane 4, 1 nM; lane 5, 5 nM; lane 6, 10 nM; lane 7, 25 nM; lane 8, 50 nM. Lanes 1–2, A and G dideoxynucleotide sequencing reactions. The extent of the interaction between Rex and cydP1, with respect to the transcription initiation site, is indicated by a vertical grey bar (see Figure 2A for sequence). (B) EMSAs using cydP1 mutant promoter fragments (see Figure 2A) and purified Rex. Only the upstream ROP half-site is disrupted in cydP1-M11, both half-site and complete ROP site are disrupted in cydP1-M12, and only the complete ROP site is disrupted in cydP1-M13. Each assay contained 1 nM promoter DNA and 25 or 200 nM Rex. The open arrow indicates unbound probe and the closed arrows indicate Rex–DNA complexes.