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Properties of RNA polymerase bypass mutants: implications for the role of ppGpp and its co-factor DksA in controlling transcription dependent on sigma54.
Umeå University, Faculty of Science and Technology, Molecular Biology (Faculty of Science and Technology). (Shingler)
Umeå University, Faculty of Science and Technology, Molecular Biology (Faculty of Science and Technology). (Shingler)
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2007 (English)In: Journal of Biological Chemistry, ISSN 0021-9258, Vol. 282, no 25, 18046-56 p.Article in journal (Refereed) Published
Abstract [en]

The bacterial nutritional and stress alarmone ppGpp and its co-factor DksA directly bind RNA polymerase to regulate its activity at certain sigma70-dependent promoters. A number of promoters that are dependent on alternative sigma-factors function poorly in the absence of ppGpp. These include the Pseudomonas-derived sigma54-dependent Po promoter and several other sigma54-promoters, the transcription from which is essentially abolished in Escherichia coli devoid of ppGpp and DksA. However, ppGpp and DksA have no apparent effect on reconstituted in vitro sigma54-transcription, which suggests an indirect mechanism of control. Here we report analysis of five hyper-suppressor mutants within the beta- and beta'-subunits of core RNA polymerase that allow high levels of transcription from the sigma54-Po promoter in the absence of ppGpp. Using in vitro transcription and competition assays, we present evidence that these core RNA polymerase mutants are defective in one or both of two properties that could combine to explain their hyper-suppressor phenotypes: (i) modulation of competitive association with sigma-factors to favor sigma54-holoenzyme formation over that with sigma70, and (ii) reduced innate stability of RNA polymerase-promoter complexes, which mimics the essential effects of ppGpp and DksA for negative regulation of stringent sigma70-promoters. Both these properties of the mutant holoenzymes support a recently proposed mechanism for regulation of sigma54-transcription that depends on the potent negative effects of ppGpp and DksA on transcription from powerful stringent sigma70-promoters, and suggests that stringent regulation is a key mechanism by which the activity of alternative sigma-factors is controlled to meet cellular requirements.

Place, publisher, year, edition, pages
2007. Vol. 282, no 25, 18046-56 p.
Keyword [en]
Binding; Competitive, DNA-Directed RNA Polymerases/*genetics/*metabolism, Escherichia coli/metabolism, Escherichia coli Proteins/*metabolism, Gene Expression Regulation; Bacterial, Models; Biological, Mutation, Promoter Regions (Genetics), Pseudomonas/metabolism, Pyrophosphatases/*physiology, RNA Polymerase Sigma 54/*metabolism, Transcription; Genetic
URN: urn:nbn:se:umu:diva-16710DOI: 10.1074/jbc.M610181200PubMedID: 17456470OAI: diva2:156383
Available from: 2009-04-16 Created: 2009-04-15 Last updated: 2010-03-04Bibliographically approved
In thesis
1. On the role of small regulatory molecules in the interplay between σ54- and σ70-dependent transcription
Open this publication in new window or tab >>On the role of small regulatory molecules in the interplay between σ54- and σ70-dependent transcription
2009 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Signal responsive transcriptional control in bacteria is mediated through both specific and global regulatory circuits to attune promoter output to prevailing conditions. Divergent transcription of a regulatory gene and a cognate promoter under its control provides an opportunity for interplay between transcription dependent on RNA polymerases utilizing various σ-factors, each of which programs the holoenzyme to recognize different classes of promoters. The work presented in this thesis analyses the consequences and mechanisms behind interplay between σ54- and σ70-dependent transcription within the dmp-system of Pseudomonas sp. CF600. The dmp-system confers the ability to grow at the expense of (methyl)phenols and is controlled by two promoters that drive non-overlapping divergent transcription from a common intergenic region: i) the σ54-Po promoter, which controls an operon encoding a suit of specialized catabolic enzymes, and ii) the σ70-Pr promoter, which controls production of the aromatic sensor DmpR - a mechano-activator whose transcription-promoting activity is obligatory for activity of the σ54-Po promoter.

The σ54-Po promoter and its dependence on two non-classical transcriptional regulators - the alarmone ppGpp and its co-factor DksA that directly target RNA polymerase - are the focus of the first part of the thesis. These studies utilized ppGpp and DksA deficient strains, mutant RNA polymerases that bypass the need for ppGpp and DksA, reconstituted in vitro transcription systems, and a series of DmpR-regulated hybrid σ54-promoters with different affinities for σ54-RNA polymerase, together with analysis of protein levels of key transcriptional components. Collectively with previous work, these studies provide the experimental support for a robust but purely passive mechanism for ppGpp and DksA global regulation of σ54-transcription, which is likely to also be pertinent for transcription mediated via any alternative σ-factor (Papers I-III). The second part of the thesis focuses on additional roles of ppGpp and DksA through their direct and indirect effects on the activity of the σ70-Pr promoter. These studies unexpectedly revealed that the σ70-Pr promoter is regulated by a novel mechanism in which σ54-RNA polymerase occupancy and activity at the σ54-Po promoter stimulates σ70-Pr output. Evidence is presented that ppGpp and DksA, through DmpR levels, control a feed forward loop to reinforce silence of the σ54-Po promoter under high energy conditions with robust transcription from σ54-Po when the catabolic enzymes are needed. The interplay outlined above effectively places a σ70-dependent promoter under dual control of two forms of RNA polymerases, and also makes it subservient to regulatory signals that elicit activity of σ54-RNA polymerase. The possibility that such dual sensitivity may be a prevalent, but previously unappreciated, mechanism by which bacteria integrate diverse and/or conflicting signals to gain appropriate transcriptional control is discussed.

36 p.
σ54, σ70, ppGpp, DksA, transcription regulation
National Category
Biochemistry and Molecular Biology
Research subject
Molecular Biology
urn:nbn:se:umu:diva-21647 (URN)978-91-7264-764-0 (ISBN)
Public defence
2009-05-08, Major Groove, Molekylärbiologi, Byggnad 6L, Umeå, 10:00 (English)
Available from: 2009-04-16 Created: 2009-04-14 Last updated: 2009-04-16Bibliographically approved

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