Figure 3

Binding of Zur to the promoter regions of its potential target genes. The [γ-³²P]-labeled upstream region of each genes (10 fmol of target DNA probes) were incubated with the purified Zur protein in the presence of 100 μM ZnCl₂. 0, 1.25, 2.5, 5, 5, 5 and 0 pmol of Zur were used in lanes 1 to 4 and C1 to C3, respectively. The mixtures were directly subjected to 4% polyacrylamide gel electrophoresis. For lanes 1 to 4, the retarded DNA band with decreased mobility turned up, which presumably represented the Zur-DNA complex. To confirm the specificity of the binding complexes, either a 200-fold molar excess of nonspecific competitor (2 pmol of unlabeled znuA DNA without its predicted binding region in lane C1) or a 200-fold molar excess of specific competitor (2 pmol of unlabeled target DNA probe in lane C2) was added to the binding mixture. 2 pmol of an unrelated protein, i.e., purified rabbit anti-F1 antibody, were included in lane C3. Both znuA and znuC gave positive EMSA results. Since these two genes had overlapped upstream regions and shared a single predicted Zur site, the EMSA data of only znuA rather than znuC was presented herein.