FIG 5.

DNase I footprinting analysis of competition of binding of CodY to CodY-I+II with that of the complex of CcpA and P-Ser-HPr to cre. The footprinting experiments were carried out as described in the text. (A) The probe DNA (wild type) was the ilv-leu promoter fragment (nt −248/+26). The gel is a DNase I footprint of the 5′-end-labeled coding strand. Binding of CcpA/P-Ser-HPr to cre and CodY to its sites was carried out in the presence of 2 mM GTP and 10 mM (each) isoleucine, valine, and leucine, with constant concentrations of CcpA (3.0 μM) and P-Ser-HPr (3.7 μM), as described in the text. Lane 1 contained no protein. Lanes 2, 3, 4, and 5 contained 0, 125, 250, and 500 nM CodY. Lanes G, A, T, and C contained the products of the corresponding sequencing reactions performed with the same primers as those used for the probe preparations. The protected areas of the cre sequence and CodY-I+II to CodY-IV sites are enclosed in boxes. (B) The probe DNA (Δ21) was the ilv-leu promoter fragment (nt −248/+26) carrying the Δ21 (nt −72/−52) deletion. The gel is a DNase I footprint of the 5′-end-labeled coding strand. The respective conditions for protection of CodY sites and the cre site by CodY and of CcpA and P-Ser-HPr are shown in panel A. The assignment of lanes (G, A, T, C, and lanes 1 to 5) was the same as that for panel A.