FIGURE 8.

Bandshifts showing the effect of citrate-Mg²⁺ on AcnR-DNA binding. A, influence of Mg²⁺ and citrate on AcnR-DNA binding, separately and in combination. A 2 nm concentration of a Cy3-labeled DNA fragment covering the promoter region of acn was incubated with AcnR, Mg²⁺, and/or citrate as indicated. Only if both citrate and Mg²⁺ are added can a backshift be observed. B, reversibility of the citrate-Mg²⁺ effect. AcnR was incubated with DNA in binding buffer. A sample was taken (2), and the remaining complex was supplemented with Mg²⁺ and citrate. After a further 5 min, another sample was taken (3). EDTA was added, and another sample was taken after 5 min (4). C, influence of citrate-Mg²⁺ on mutational variants of AcnR. Arg-130 is involved in citrate binding, and the R130A variant no longer reacts to citrate-Mg²⁺. The same amino acid exchange at another position (Arg-92 within the P pocket) does not change the behavior of AcnR in the presence of citrate-Mg²⁺.