Figure 6
Zur protein binds to the hly II promoter. (a) Sequence of the hly II operon upstream of hlyC. In bold are indicated the first codon (ATG) of hlyC and the putative −10 and −35 boxes of the promoter. Within the blue box is indicated the putative binding site of Zur. The red box indicates the transcriptional start found by 5′RACE assays. (b) Sequence of the putative Zur binding site found in the hly II operon promoter. Bases recognized by Zur dimers are colored: green for conserved bases and red for the only base that is not conserved. A dashed line represents the symmetry axis. Below are depicted the positions of Zur binding sites in promoters of znuABC, zinT, rpmE29 and hlyII. Zur boxes were drawn to scale in respect of transcription start site. (c) Sequence of the 5′RACE assay using J96 total RNA samples grown in LB at 37 °C up to late log phase. The two bases identified as a transcriptional start are indicated with arrowheads. The N residue corresponds to a mix of A and C bases. (d) Electrophoretic mobility shift assay. Titration of a Cy5 labeled 50-bp hly II promoter fragment carrying the putative Zur binding site was carried out in the presence of excess salmon sperm DNA which serves as a non-specific competitor. Samples contains 60 pM DNA, plus 0, 15, 30, 50, 100, 150, 200, 300, 350, 900 pM Zur (calculated as dimer), respectively. Samples were resolved on a 10% polyacrylamide gel. Both gel and electrophoresis buffers contain 50 µM ZnSO4. Full-length gel image is shown in Fig. S9.
