Figure 4.

MisR binding sites located upstream of the dsbD promoter. (A) DNase I protection. The coding (left) or noncoding (right) strand of ³²P-end labeled dsbD promoter fragment between −227 to +123 with respect to the dsbD transcriptional start site was incubated with increasing amounts of MisR for 20 minutes at 30°C and then subjected to DNase I digestion. The amounts of MisR used in the left panel are 0, 40, 80, and 120 pmol, while in the right panel are 0, 40, 80, 120, 200 and 0 pmol. The dideoxy chain-termination sequencing ladders were generated by extension of ³²p-labeled pF2 primer for the coding strand or pR1 primer for the noncoding strand using the corresponding PCR product as template. Black bars indicate protected regions. (B) The dsbD promoter sequence. The protected regions are indicated as shaded boxes in both strands. The transcriptional start site +1, −10 and −35 promoter elements are bolded and underlined. The predicted MisR binding motifs are italicized and marked with dashed lines above the sequence. Bent arrows indicate the 5′ ends of various promoter fragments cloned in reporter strains.