FIGURE 3.
The interaction of SlyA with the fimB promoter region in vitro. A, effect of 0, 15, 30, 60, and 120 nm SlyA dimer on the electophoretic mobility of DNA amplicons fim03 and pBS (each 11 nm). Amplicon fim03 is 282 bp of the region upstream of fimB and includes OSA1 and OSA2 (Fig. 1). Amplicon pBS is a negative control as previously described (27). The SlyA-DNA complex is indicated with an arrow. Samples were separated on a 5% polyacrylamide gel. Electrophoresis was carried out at 160 V for 35 min as described under “Experimental Procedures.” B, DNase I footprinting. The fim03 fragment, labeled at the fim03r end, was mixed with decreasing concentrations of SlyA for 15 min at 25 °C before digestion with DNaseI. Lane 1, no SlyA; lane 2, 1 μm SlyA; lane 3, 500 nm; lane 4, 250 nm; lane 5, 125 nm; lane 6, 62.5 nm; lane 7, 31 nm; lane 8, 15.6 nm. The products were analyzed on a 6% denaturing polyacrylamide gel. Regions protected by SlyA are indicated. The marker is pBR322 digested with MspI, and the sizes of the fragments were used to calculate their positions relative to the transcriptional start site of fimB (+1).