Figure 3.

EMSA analysis of the stoichiometrical requirement of IdeR to bind its target DNA. (A) Binding of IdeR to a DNA sequence designed to bind only one IdeR dimer. (B) Binding of IdeR to the full consensus DNA sequence and the designed half binding site. (C) Binding of IdeR to site 1 of cluster 23. (D) Binding of IdeR to the full consensus DNA sequence and site 1 of cluster 23. IdeR was added to 30 nM fluorescence-labeled double-stranded DNA probe in (A, C) increasing concentrations (15 nM–2.25 μM dimer) or (B, D) at 1.5 μM in the presence of (A, C) 30 μM or (B, D) 40 μM Co²⁺ and competitor DNA. Protein–DNA complexes were resolved on a 4% Tris-acetate polyacrylamide gel. The left-most lane in panels A and C is a control reaction without protein. The gel images in panels B and D have been edited for easier comparison between both samples. ds, unbound double-stranded DNA probe; ss, non-hybridized single-stranded DNA; C, protein–dsDNA complex; 2C, protein–dsDNA complex containing two IdeR dimers; 1C, protein–dsDNA complex containing one IdeR dimer.