Abstract
The interactions between the sigma54-containing RNA polymerase (sigma54-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 sigma54-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 sigma54-RNAP in vitro. The experiments with oriented-alpha sigma54-RNAP derivatives containing bound chemical nuclease revealed interchangeable positioning of only one of the two alpha subunit carboxyl-terminal domains (alphaCTDs) both at the -104 region and in the surroundings of position -78. The addition of IHF resulted in perfect position symmetry of the two alphaCTDs. These results indicate that, in the absence of IHF, the sigma54-RNAP asymmetrically uses only one alphaCTD 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 sigma54-RNAP can allow the other alphaCTD to be engaged in and thus favor closed complex formation.
Highlights
Induced curvature, the closer proximity of the upstream DNA to the body of the 54-RNAP can allow the other ␣CTD to be engaged in and favor closed complex formation
The bending activity of integration host factor (IHF) strongly augments the probability of interaction between the activator XylR and 54-RNAP [44] and positively influences the docking of 54-RNAP on the promoter [26]
Our results suggested that the upstream interactions of 54RNAP by ␣CTD could play an active role in its IHF-mediated recruitment
Summary
Induced curvature, the closer proximity of the upstream DNA to the body of the 54-RNAP can allow the other ␣CTD to be engaged in and favor closed complex formation. The ability of ␣CTD to contact DNA and/or activator molecules at different locations upstream of the core promoter [8, 15,16,17,18,19,20,21] has been attributed to the flexibility of the linker connecting ␣CTD to the ␣ amino-terminal domain (␣NTD) [8, 22] assembled in the body of RNAP This linker flexibility accounts for the ability of the two copies of ␣CTD to function interchangeably with respect to the subsite recognition within the UP element [10]. This paper is available on line at http://www.jbc.org
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