FIG. 3.

Gel retardation assays with the nrf promoter. The figure shows gel retardation assays with pnrf53 promoter fragments incubated with purified IHF (A) and Fis (B) proteins. (A) ³²P-end-labeled pnrf53 EcoRI-MfeI fragments were incubated with increasing concentrations of purified IHF protein, as follows: lanes 1 to 5, wild-type pnrf53; lanes 6 to 10, pnrf53/p104Gp103C (IHF II); and lanes 11 to 15, pnrf53/p124Gp123C (IHF III). The concentrations of IHF protein in the reactions were as follows: lanes 1, 6, and 11, no protein; lanes 2, 7, and 12, 25 nM; lanes 3, 8, and 13, 50 nM; lanes 4, 9, and 14, 0.1 μM; and lanes 5, 10, and 15, 0.2 μM. (B) ³²P-end-labeled pnrf53 EcoRI-MfeI fragments were incubated with increasing concentrations of purified Fis protein, as follows: lanes 1 to 4, wild-type pnrf53; and lanes 5 to 8, pnrf53/p175G (Fis II). The concentrations of Fis protein in the reactions were as follows: lanes 1 and 5, no protein; lanes 2 and 6, 0.11 μM; lanes 3 and 7, 0.22 μM; and lanes 4 and 8, 0.44 μM. Note that pnrf53 EcoRI-MfeI fragments carry pnrf sequences from positions −209 to −50 and, therefore, only carry the IHF II, IHF III, and Fis II binding sites.