FIG. 3.
Quantitative DNase I footprinting of DNA fragments bearing mutated araABDLMNPQ-abfA operon promoters. DNA fragments of 263 and 269 bp carrying the ara metabolic operon promoter region with 5-bp (A) and 11-bp (B) insertions, respectively, between ORA1 and ORA2 were end labeled with [γ-32P]ATP and used as target DNA in the assays. Only the coding strand was radiolabeled, as described in Materials and Methods. AraR concentrations were calculated by reference to a pure dimeric protein. Lane 1, no protein; lane 2, 25 nM AraR; lane 3, 50 nM AraR; lane 4, 100 nM AraR; lane 5, 150 nM AraR; lane 6, 200 nM AraR; lane 7, 250 nM AraR; lane 8, 250 nM AraR plus 15 mM l-arabinose. DNA sequencing reactions were run side by side with the DNase I footprinting reactions (not shown), and the localization of the AraR binding sites in the wild-type promoter (ORA1 and ORA2) are indicated in the auradiographs by brackets. A schematic representation of the DNA fragments used in the experiments, with the DNA insertions made between ORA1 and ORA2 represented by white triangles, is shown above the autoradiographs: AraR binding sites are represented as gray boxes, DNA from the araABDLMNPQ-abfA operon promoter is shown as white boxes, and heterologous DNA (from plasmid pSN32 [see Materials and Methods]) is shown as dotted boxes. The total repressor concentration at which the half-maximal site occupancy is achieved (a value that represents the apparent affinity of AraR for each site) is indicated within parentheses for each operator in the two DNA fragments.