Figure 4

Footprint analysis of the sacB promoter. A. To analyze the top and bottom strands, the oligonucleotide pairs sacB-bio3 and sacB-D, and sacB-U and sacB-Pex2 were used for PCR amplification, respectively. The sequencing ladder template was generated by PCR using the oligonucleotide pair sacB-seq and sacB-U. The ladders were generated by using the primer sacB-bio3 and sacB-Pex2. The probe (40 nM) was 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. Lane 1. No protein; 4, 0.15 μM of DegU and 0.05 μM His-tagged DegS; 2 and 5, 0.3 μM of DegU and 0.1 μM His-tagged DegS; 3 and 6; 0.6 μM of DegU and 0.2 μM His-tagged DegS. 2 and 3, no ATP; 4, 5 and 6, 1 mM ATP. The nucleotide sequence of the promoter region is shown. The solid and dotted brackets indicate the strongly and weakly protected regions, respectively. B. Arrows above the sequences and the numbers indicate the DegU-binding motifs and the nucleotide positions relative to the transcription start site, respectively.