FIG 6.
Specific binding of CRP to the PrtxA regulatory region. (A and B) Each 452-bp DNA probe of the PrtxA regulatory region (probe 1 for panel A and probe 2 for panel B; 5 nM) was radioactively labeled and then incubated with increasing amounts of CRP as indicated. For competition analysis, various amounts of the same but unlabeled DNA fragment were used as a self-competitor and added to the reaction mixture containing the 5 nM labeled DNA before the addition of 600 nM (A) or 400 nM (B) CRP. The DNA-protein complexes were separated by electrophoresis. B, bound DNA; F, free DNA. (C and D) The same DNA probes of each PrtxA regulatory region (40 nM) were labeled with 6-FAM, incubated with increasing amounts of CRP as indicated, and then digested with DNase I. The regions protected by CRP are indicated by gray boxes (CRPB1, CRPB2, and CRPB3), respectively. The nucleotides showing enhanced cleavage are indicated by asterisks. Nucleotide numbers shown are relative to the transcription start site of rtxA.