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The guanosine tetraphosphate (ppGpp) alarmone, DksA and promoter affinity for RNA polymerase in regulation of σ54-dependent transcription
Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology). (Shingler)
Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology). (Shingler)
Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology). (Shingler)
Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology). (Shingler)
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2006 (English)In: Molecular Microbiology, ISSN 0950-382X, E-ISSN 1365-2958, Vol. 60, no 3, 749-764 p.Article in journal (Refereed) Published
Abstract [en]

The RNA polymerase-binding protein DksA is a cofactor required for guanosine tetraphosphate (ppGpp)-responsive control of transcription from sigma70 promoters. Here we present evidence: (i) that both DksA and ppGpp are required for in vivo sigma54 transcription even though they do not have any major direct effects on sigma54 transcription in reconstituted in vitro transcription and sigma-factor competition assays, (ii) that previously defined mutations rendering the housekeeping sigma70 less effective at competing with sigma54 for limiting amounts of core RNA polymerase similarly suppress the requirement for DksA and ppGpp in vivo and (iii) that the extent to which ppGpp and DksA affect transcription from sigma54 promoters in vivo reflects the innate affinity of the promoters for sigma54-RNA polymerase holoenzyme in vitro. Based on these findings, we propose a passive model for ppGpp/DksA regulation of sigma54-dependent transcription that depends on the potent negative effects of these regulatory molecules on transcription from powerful stringently regulated sigma70 promoters.

Place, publisher, year, edition, pages
2006. Vol. 60, no 3, 749-764 p.
Keyword [en]
DNA-Directed RNA Polymerases/genetics/*metabolism, Escherichia coli/genetics/*metabolism, Escherichia coli Proteins/genetics/*metabolism, Gene Expression Regulation; Bacterial, Guanosine Tetraphosphate/*metabolism, Mutation, Promoter Regions (Genetics), RNA Polymerase Sigma 54/genetics/*metabolism, Sigma Factor/genetics/metabolism, Transcription; Genetic
Identifiers
URN: urn:nbn:se:umu:diva-16715DOI: 10.1111/j.1365-2958.2006.05129.xPubMedID: 16629675OAI: oai:DiVA.org:umu-16715DiVA: diva2:156388
Available from: 2009-04-15 Created: 2009-04-15 Last updated: 2017-05-24Bibliographically approved
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.
Keyword
ppGpp, DksA, σ54-transcription, global regulation, E. coli, P. putida
National Category
Biochemistry and Molecular Biology
Identifiers
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)
Opponent
Supervisors
Available from: 2006-11-16 Created: 2006-11-16 Last updated: 2017-05-24Bibliographically approved
2. 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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Skärfstad, EleonoreShingler, Victoria

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