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σ54-promoter discrimination and regulation by ppGpp and DksA
Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology). (Victoria Shingler)
Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
2009 (English)In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 284, no 2, 828-838 p.Article in journal (Refereed) Published
Abstract [en]

The sigma(54)-factor controls expression of a variety of genes in response to environmental cues. Much previous work has implicated the nucleotide alarmone ppGpp and its co-factor DksA in control of sigma(54)-dependent transcription in the gut commensal Escherichia coli, which has evolved to live under very different environmental conditions than Pseudomonas putida. Here we compared ppGpp/DksA mediated control of sigma(54)-dependent transcription in these two organisms. Our in vivo experiments employed P. putida mutants and manipulations of factors implicated in ppGpp/DksA mediated control of sigma(54)-dependent transcription in combination with a series of sigma(54)-promoters with graded affinities for sigma(54)-RNA polymerase. For in vitro analysis we used a P. putida-based reconstituted sigma(54)-transcription assay system in conjunction with DNA-binding plasmon resonance analysis of native and heterologous sigma(54)-RNA polymerase holoenzymes. In comparison with E. coli, ppGpp/DksA responsive sigma(54)-transcription in the environmentally adaptable P. putida was found to be more robust under low energy conditions that occur upon nutrient depletion. The mechanism behind this difference can be traced to reduced promoter discrimination of low affinity sigma(54)-promoters that is conferred by the strong DNA binding properties of the P. putida sigma(54)-RNA polymerase holoenzyme.

Place, publisher, year, edition, pages
Bethesda: The American Society for Biochemistry and Molecular Biology Inc , 2009. Vol. 284, no 2, 828-838 p.
National Category
Biochemistry and Molecular Biology
Research subject
Molecular Biology
Identifiers
URN: urn:nbn:se:umu:diva-21643DOI: 10.1074/jbc.M807707200OAI: oai:DiVA.org:umu-21643DiVA: diva2:211405
Available from: 2009-04-15 Created: 2009-04-14 Last updated: 2017-12-13Bibliographically 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.

Publisher
36 p.
Keyword
σ54, σ70, ppGpp, DksA, transcription regulation
National Category
Biochemistry and Molecular Biology
Research subject
Molecular Biology
Identifiers
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)
Opponent
Supervisors
Available from: 2009-04-16 Created: 2009-04-14 Last updated: 2009-04-16Bibliographically approved

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Johansson, Linda U MSkärfstad, EleonoreShingler, Victoria
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