Fur recognizes a Fur box within the hlyII promoter and inhibits hlyII transcription in vitro by competing with RNA polymerase. (A) DNase I footprint analyses for the noncoding strand (template strand) of the hlyII promoter. Lane 1, product of G/A sequencing reaction. Lanes 2 to 7, results from DNase I footprinting. Lanes 2 and 7, empty DNA; lane 3, no RNAP, 1.4 μM Fur; lane 4, 0.1 μM RNAP, no Fur; lane 5, 1.4 μM Fur added first and then 0.1 μM RNAP; lane 6, 0.1 μM RNAP and then 1.4 μM Fur. (B) Runoff product formation. (C) Abortive initiation. (D) Time-dependent synthesis of runoff product in the presence of 0.25 μM Fur. (E) Fur and HlyIIR may work together in transcription inhibition. Fifty nanograms of 400-bp PhlyII fragments and proteins was combined in the presence of GTP, ATP, CTP, and [α-32P]UTP. Lane 1, 0.1 μM RNAP; lanes 2 and 3, 2 and 4 μM HlyIIR, respectively, and then 0.1 μM RNAP; lanes 4 and 5, 0.7 and 1.4 μM Fur, respectively, and then 0.1 μM RNAP; lanes 6 and 7, 0.1 μM RNAP and then 0.7 and 1.4 μM Fur, respectively; lanes 8 and 9, 2 and 4 μM HlyIIR, respectively, 0.1 μM RNAP, and 0.7 μM Fur added last. (F) EDTA can reduce the effect of Fur inhibition. Protein concentrations are as in Fig. 5E.