FIG 5.
Characterization of the HutC-binding site in the ntrBC promoter region. (A) DNase I footprinting was performed using the PntrBC-154 probe labeled at the 3′ end. Lane M, G+A marker; lane 6, no HutCHis6; lanes 1 to 5, HutCHis6 added at 0.26, 0.77, 1.8, 3.34 and 5.14 μM, respectively. (B) DNase I analysis showing the effect of urocanate on HutCHis6 binding. Lane 1, no HutCHis6 and no urocanate; lanes 2 to 5, HutCHis6 (5.14 μM) with urocanate added at 0, 0.2, 0.5, and 2.0 mM, respectively. (C) EMSA of HutCHis6 using 3′-end biotin-labeled probe PntrBC-154 or mutant probe PntrBC-mut2 carrying mutations in the Phut motif. Lanes 1 to 4, HutCHis6 added at 0, 0.64, 1.28, and 1.92 μM, respectively. The position of free probe is marked by an asterisk. (D) Sequence of the HutC-protected region as revealed by DNase I footprinting. The two HutC-binding half sites are shown by underlined letters, and the corresponding sequence was mutated in the PntrBC-mut2 probe DNA.