Recruitment of σ⁵⁴-RNA polymerase to the Pu promoter of Pseudomonas putida through integration host factor-mediated positioning switch of α subunit carboxyl-terminal domain on an UP-like element

The interactions between the σ⁵⁴-containing RNA polymerase (σ⁵⁴-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 σ⁵⁴-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 σ⁵⁴-RNAP in vitro. The experiments with oriented-α σ⁵⁴-RNAP derivatives containing bound chemical nuclease revealed interchangeable positioning of only one of the two α subunit carboxyl-terminal domains (αCTDs) both at the -104 region and in the surroundings of position -78. The addition of IHF resulted in perfect position symmetry of the two αCTDs. These results indicate that, in the absence of IHF, the σ⁵⁴-RNAP asymmetrically uses only one αCTD 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 σ⁵⁴-RNAP can allow the other αCTD to be engaged in and thus favor closed complex formation.