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 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.
Macchi
Raffaella
R
Dipartimento di Genetica e Biologia dei Microrganismi, Università degli Studi di Milano, via Celoria 26, 20133 Milan, Italy.
Montesissa
Lorena
L
Murakami
Katsuhiko
K
Ishihama
Akira
A
De Lorenzo
Victor
V
Bertoni
Giovanni
G
D001483:Base Sequence; D001665:Binding Sites; D004268:DNA-Binding Proteins; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism*; D003850:Deoxyribonuclease I / Q000378:metabolism; D017353:Gene Deletion; D017665:Hydroxyl Radical / Q000378:metabolism; D008954:Models, Biological; D008969:Molecular Sequence Data; D010957:Plasmids / Q000378:metabolism; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D016958:Pseudomonas putida / Q000235:genetics*; D051877:RNA Polymerase Sigma 54; D012808:Sigma Factor / Q000378:metabolism*; D013997:Time Factors
D004268:DNA-Binding Proteins; D012808:Sigma Factor; D017665:Hydroxyl Radical; D012321:DNA-Directed RNA Polymerases; D051877:RNA Polymerase Sigma 54; D003850:Deoxyribonuclease I