Figure 3.

Footprint of NagC and Mlc binding to the Nag14E–Nag15B fragment labelled at Nag15B (A) or Nag14E (B). The DNA was incubated with extracts containing overproduced NagC (lanes A3 and B4), Mlc (lanes A2 and B2) or an empty plasmid vector control (lane A4) (~100 µg/ml) in binding buffer, 50 mM HEPES, 100 mM Na glutamate, pH 8.0, 0.5 mg/ml BSA. Lanes labelled with an F show DNA in the absence of protein extracts. After DNaseI digestion, the reactions were extracted with phenol and analysed on an 8% denaturing polyacylamide gel as described previously (22). The nagE and nagB operators (BoxE and BoxB) are indicated as are the seven hypersensitive cleavages produced by NagC binding. (C) The organisation of the nagE-B promoter region with the location of the NagC operators (BoxE and BoxB) and the CAP site. The sequence is numbered from the nagE transcription site (+1) so that the nagB transcription start is at –133. The 5′ ends of the oligonucleotides are indicated by an asterisk and are at +46 for Nag14E, –118 for Nag42B and –187 for Nag15B. HphI cuts at –56/57 and the XbaI site was created by insertion of 6 bp at –91.