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The role of the alarmone (p)ppGpp in sigma N competition for core RNA polymerase.
Umeå University, Faculty of Medicine, Molecular Biology.
Umeå University, Faculty of Medicine, Molecular Biology.
Umeå University, Faculty of Medicine, Molecular Biology.
Umeå University, Faculty of Medicine, Molecular Biology.
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2003 (English)In: Journal of Biological Chemistry, ISSN 0021-9258, Vol. 278, no 3, 1494-1503 p.Article in journal (Refereed) Published
Abstract [en]

Some promoters, including the DmpR-controlled sigma(N)-dependent Po promoter, are effectively rendered silent in cells lacking the nutritional alarmone (p)ppGpp. Here we demonstrate that four mutations within the housekeeping sigma(D)-factor can restore sigma(N)-dependent Po transcription in the absence of (p)ppGpp. Using both in vitro and in vivo transcription competition assays, we show that all the four sigma(D) mutant proteins are defective in their ability to compete with sigma(N) for available core RNA polymerase and that the magnitude of the defect reflects the hierarchy of restoration of transcription from Po in (p)ppGpp-deficient cells. Consistently, underproduction of sigma(D) or overproduction of the anti-sigma(D) protein Rsd were also found to allow (p)ppGpp-independent transcription from the sigma(N)-Po promoter. Together with data from the direct effects of (p)ppGpp on sigma(N)-dependent Po transcription and sigma-factor competition, the results support a model in which (p)ppGpp serves as a master global regulator of transcription by differentially modulating alternative sigma-factor competition to adapt to changing cellular nutritional demands.

Place, publisher, year, edition, pages
2003. Vol. 278, no 3, 1494-1503 p.
URN: urn:nbn:se:umu:diva-26210DOI: 10.1074/jbc.M209268200PubMedID: 12421818OAI: diva2:240821
Available from: 2009-09-30 Created: 2009-09-30 Last updated: 2010-03-04
In thesis
1. On the role of ppGpp and DksA mediated control of σ54-dependent transcription
Open this publication in new window or tab >>On the role of ppGpp and DksA mediated control of σ54-dependent transcription
2006 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The σ54-dependent Po promoter drives transcription of an operon that encodes a suite of enzymes for (methyl)phenols catabolism. Transcription from Po is controlled by the sensor-activator DmpR that binds (methyl)phenol effectors to take up its active form. The σ54 factor imposes kinetic constraints on transcriptional initiation by the σ54-RNA polymerase holoenzyme which cannot undergo transition from the closed complex without the aid of the activator. DmpR acts from a distance on promoter-bound σ54-holoenzyme, and physical contact between the two players is facilitated by the DNA-bending protein IHF. The bacterial alarmone ppGpp and DksA directly bind RNA polymerase to have far reaching consequences on global transcriptional capacity in the cell. The work presented in this thesis uses the DmpR-regulated Po promoter as a framework to dissect how these two regulatory molecules act in vivo to control the functioning of σ54-dependent transcription. The strategies employed involved development of i) a series of hybrid σ54-promoters that could be directly compared and in which key DNA elements could be manipulated ii) mutants incapable of synthesizing ppGpp and/or DksA, iii) reconstituted in vitro transcription systems, and iv) genetic selection and purification of mutant RNA polymerases that bypass the need for ppGpp and DksA in vivo. The collective results presented show that the effects of ppGpp and DksA on σ54-dependent transcription are major, with simultaneous loss of these regulatory molecules essentially abolishing σ54-transcription in intact cells. However, neither of these regulatory molecules have discernable effects on in vitro reconstituted σ54-transcription, suggesting an indirect mechanism of control. The major effects of ppGpp and DksA in vivo cannot be accounted for by consequent changes in the levels of DmpR or other specific proteins needed for σ54-transcription. The data presented here shows i) that the effects of loss of ppGpp and DksA are related to promoter affinity for σ54-holoenzyme, ii) that σ54 is under significant competition with other σ-factors in the cell, and iii) that mutants of σ70, and the beta- and beta prime-subunits of RNA polymerase that can bypass the need for ppGpp and DksA in vivo have defects that would favour the formation of σ54-RNA holoenzyme over that with σ70, and that mimic the effects of ppGpp and DksA for negative regulation of stringent σ70-promoters. A purely passive model for ppGpp/DksA regulation of σ54-dependent transcription that functions through their potent negative effects on transcription from powerful σ70-stringent promoters is presented.

Place, publisher, year, edition, pages
Umeå: Molekylärbiologi (Teknisk-naturvetenskaplig fakultet), 2006. 69 p.
ppGpp, DksA, σ54-transcription, global regulation, E. coli, P. putida
National Category
Biochemistry and Molecular Biology
urn:nbn:se:umu:diva-932 (URN)91-7264-217-3 (ISBN)
Public defence
2006-12-07, Major Groove, 6L, Institutionen för Molekylärbiologi, Umeå Universitet, 90187, Umeå, 09:30 (English)
Available from: 2006-11-16 Created: 2006-11-16 Last updated: 2009-09-30Bibliographically approved

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