Fig. 2.

PrpR interacts with the dnaA promoter region in vitro and in vivo. A EMSA: a DNA fragment containing the dnaA promoter region (PCR amplified with primers pmtdnaA_Fw2 and MtrpmH_Rv, Table 1) was incubated with a non-specific competitor and increasing amounts of 6HisPrpR, and the nucleoprotein complexes were analyzed on a 4 % polyacrylamide gel; prpDR (containing the perfect PrpR biding site) and the mtrA promoter region served as positive and negative controls, respectively. Arrows along horizontally orientated bars inserted below each figure indicate number of PrpR boxes in corresponding DNA fragment. Digits above arrows indicate number of nucleotide mismatches from the consensus (TTTGCAAA) within each PrpR box. B In vivo immunoprecipitation: PrpR–DNA complexes cross-linked with glutaraldehyde were immunoprecipitated with anti-6HisPrpR polyclonal antibodies (sample 1). PCR was carried out with the following primer pairs: p1129_Fw and p1129_Rv (pprpDR, positive control); pmtdnaA_Fw2 and MtrpmH_Rv (pdnaA); and pmtrA_Fw and pmtrA_Rv (pmtrA, negative control). A second negative control (2) consisted of extracted DNA subjected to immunoprecipitation without cross-linking. Positives controls (+) were also performed using templates obtained from strains subjected only to cross-linking (3) or total DNA extracted from the cells (4)