Recruitment of sigma54-RNA polymerase to the Pu promoter of Pseudomonas putida through integration host factor-mediated positioning switch of alpha subunit carboxyl-terminal domain on an UP-like element

The interactions between the {sigma}54-containing RNA polymerase ({sigma}54-RNAP) and the region of the Pseudomonas putida Pu promoter spanning from the enhancer to the binding site for the integration host factor (IHF) were analyzed both by DNase I and hydroxyl radical footprinting. A short Pu region centered at position –104 was found to be involved in the interaction with {sigma}54-RNAP, both in the absence and in the presence of IHF protein. Deletion or scrambling of the –104 region strongly reduced promoter affinity in vitro and promoter activity in vivo, respectively. The reduction in promoter affinity coincided with the loss of IHF-mediated recruitment of the {sigma}54-RNAP in vitro. The experiments with oriented-{alpha} {sigma}54-RNAP derivatives containing bound chemical nuclease revealed interchangeable positioning of only one of the two {alpha} subunit carboxyl-terminal domains ({alpha}CTDs) both at the –104 region and in the surroundings of position –78. The addition of IHF resulted in perfect position symmetry of the two {alpha}CTDs. These results indicate that, in the absence of IHF, the {sigma}54-RNAP asymmetrically uses only one {alpha}CTD subunit to establish productive contacts with upstream sequences of the Pu promoter. In the presence of IHF-induced curvature, the closer proximity of the upstream DNA to the body of the {sigma}54-RNAP can allow the other {alpha}CTD to be engaged in and thus favor closed complex formation.