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 language | English (US) |
|---|---|
| Pages (from-to) | 27695-27702 |
| Number of pages | 8 |
| Journal | Journal of Biological Chemistry |
| Volume | 278 |
| Issue number | 30 |
| DOIs | |
| State | Published - Jul 25 2003 |
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All Science Journal Classification (ASJC) codes
- Biochemistry
- Molecular Biology
- Cell Biology
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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 journal › Article
TY - JOUR
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
PY - 2003/7/25
Y1 - 2003/7/25
N2 - 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.
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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U2 - 10.1074/jbc.M303031200
DO - 10.1074/jbc.M303031200
M3 - Article
C2 - 12754257
AN - SCOPUS:0042346380
VL - 278
SP - 27695
EP - 27702
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
SN - 0021-9258
IS - 30
ER -