Figure 8

Footprint analysis of the sacB promoter carrying IR. To analyze the top strand, the oligonucleotide pair sacB-bio3 and sacB-D was used for PCR amplification from the plasmid carrying the SC-IR4 fusion (figure 7). The procedures for sequencing ladder generation and footprint analysis were the same as those in figure 5. 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; 2, 0.15 μM of DegU and 0.05 μM His-tagged DegS; 3, 0.3 μM of DegU and 0.1 μM His-tagged DegS; 4 and 5; 0.6 μM of DegU and 0.2 μM His-tagged DegS. 2, 3 and 4, no ATP; 5, 1 mM ATP. The nucleotide sequence of the promoter region is shown. The bracket indicates the protected region. Arrows above the sequences and the numbers indicate the DegU-binding motifs and the nucleotide positions relative to the transcription start site, respectively.