Figure 6.

Crp binding to selected promoters by EMSA. (a) Interaction of the dms promoter DNA with S. oneidensis His-tagged Crp. The probe was prepared by PCR with ³³P end-labeled primers. The EMSA assay was performed with 2 nM ³³P end-labeled probes and various amounts of Crp (left panel) or Crp and cAMP (right panel). The protein concentrations for lanes 1–5 are 0, 0.25, 0.5, 1.0, 2.0 μM, respectively. Non-specific competitor DNA (0.2 μg poly dI·dC) was added to all lanes and specific competitor (10 μM unlabeled dms probe) was added (lane 6). (b) The binding assay was performed in the presence of 0, 0.5 or 2 μM Crp, 10 μM cAMP, and 2–5 nM radiolabeled promoter DNA. 0.2 μg μl⁻¹ poly(dI·dC) was used in all these binding reactions to block nonspecific interactions. Promoter region of gyrB was used as negative control.