HapR and CRP co-operatively bind the same section of murQP regulatory DNA.
a. Binding locations of HapR and CRP upstream of murQP. The gel shows the result of DNase I footprinting experiment. The gel is calibrated with Sanger sequencing reactions. The pattern of DNase I cleavage in the absence of any proteins is in lane 1. Protection of DNA from DNase I cleavage in the presence of 0.11, 0.23 or 0.45 μM CRP is shown in lanes 2-4. Sites of DNAse I hypersensitivity due to CRP binding are indicated by orange triangles. Protection from DNase I cleavage in the presence of 0.5, 1.0, 2.0 or 3.0 μM HapR is shown in lanes 5-8. Protection from DNase I cleavage, dependent on HapR, is shown by a green bar. A DNAse I hypersensitive band, unique to reactions with HapR, is shown by a green triangle. In the presence of 0.45 μM CRP, increasing concentrations of HapR result in a different DNAse I cleavage pattern, including the appearance of a different site of hypersensitivity (black triangle).
b. Binding of HapR and CRP upstream of murQP is co-operative. Electrophoretic mobility shift assays showing migration of the murQP regulatory region with different combinations of CRP (0.025, 0.05, 0.1 or 0.2 μM) and HapR (0.5, 1.0, 2.0, 3.0 or 4.0 μM). For incubations with both factors, the same range of HapR concentrations was used with 0.2 μM CRP.
c. Co-operative binding of CRP requires the shared HapR and CRP binding site. Results of an electrophoretic mobility shift assay, using the wild type murQP regulatory region or a derivative with two point mutations in the shared recognition sequence, for HapR (4.0 μM) and CRP (0.1 μM). Positions of mutations are shown in Figure 3b.
d. HapR blocks CRP mediated activation of the murQP promoter in vitro. The gel image shows the result of an in vitro transcription assay. The DNA template was plasmid pSR carrying the murQP regulatory region. Experiments were done with 0.4 μM RNA polymerase, with or without 0.05, 0.1, 0.2 or 0.5 μM CRP 0.5, 1.0, 2.0 or 3.0 μM HapR, as indicated. The RNAI transcript is plasmid-derived and acts as an internal control.
e. HapR represses CRP mediated activation of the murQP promoter in vivo. β-galactosidase activity was measured in cell lysates taken from Vibrio cholerae E7946 (solid green bars), ΔhapR derivative (open green bars), Δcrp variant (open orange bars), or cells lacking both factors (orange outline with green patterned fill). Standard deviation is shown for three independent biological replicates. Cells were grown in LB medium.