Figure 1

Footprint analysis of the flgB promoter. A. The probes were prepared by PCR amplification. To analyze the upstream (-120 to +38, the bottom strand in the left panel) and downstream (+15 to +264, the top strand in the right panel) regions, the oligonucleotide pairs flgB-F and flgB-D-bio, and flgB-DU-bio and flgB-DD, respectively, were used. The sequencing ladder templates for the upstream and downstream region analysis were generated by PCR using the oligonucleotide pairs flgB-F and flgB-D, and flgB-DU and flgB-DD, respectively. The probes (40 nM) were incubated with increasing amounts of DegU and His-DegS in the presence or absence of ATP, and subjected to DNase I cleavage. The sequencing ladder is shown in lanes G, A, T and C. The brackets and arrowheads indicate the protected regions and hypersensitive sites, respectively. 1. No protein. 2 and 4, 0.15 μM of DegU and 0.05 μM His-tagged DegS; 3 and 5, 0.3 μM of DegU and 0.1 μM His-tagged DegS. 2 and 3, no ATP; 4 and 5, 1 mM ATP. B. The nucleotide sequences of the regulatory regions are shown. The numbers indicate the nucleotide positions relative to the transcription start site. Dotted arrows above the sequences indicate the putative DegU-binding motifs.