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

Raffaella Macchi, Lorena Montesissa, Katsuhiko Murakami, Akira Ishihama, Victor De Lorenzo, Giovanni Bertoni

Research output: Contribution to journalArticle

23 Citations (Scopus)

Abstract

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.

Original languageEnglish (US)
Pages (from-to)27695-27702
Number of pages8
JournalJournal of Biological Chemistry
Volume278
Issue number30
DOIs
StatePublished - Jul 25 2003

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Integration Host Factors
Pseudomonas putida
DNA-Directed RNA Polymerases
Switches
Deoxyribonuclease I
Hydroxyl Radical
Binding Sites
Derivatives
DNA

All Science Journal Classification (ASJC) codes

  • Biochemistry

Cite this

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title = "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",
abstract = "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.",
author = "Raffaella Macchi and Lorena Montesissa and Katsuhiko Murakami and Akira Ishihama and {De Lorenzo}, Victor and Giovanni Bertoni",
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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. / Macchi, Raffaella; Montesissa, Lorena; Murakami, Katsuhiko; Ishihama, Akira; De Lorenzo, Victor; Bertoni, Giovanni.

In: Journal of Biological Chemistry, Vol. 278, No. 30, 25.07.2003, p. 27695-27702.

Research output: Contribution to journalArticle

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T1 - 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

AU - Macchi, Raffaella

AU - Montesissa, Lorena

AU - Murakami, Katsuhiko

AU - Ishihama, Akira

AU - De Lorenzo, Victor

AU - Bertoni, Giovanni

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AB - 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.

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