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On the role of ppGpp and DksA mediated control of σ54-dependent transcription
Umeå University, Faculty of Medicine, Molecular Biology.
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 [en]
ppGpp, DksA, σ54-transcription, global regulation, E. coli, P. putida
National Category
Biochemistry and Molecular Biology
Identifiers
URN: urn:nbn:se:umu:diva-932ISBN: 91-7264-217-3 (print)OAI: oai:DiVA.org:umu-932DiVA: diva2:145100
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
List of papers
1. Integration of global regulation of two aromatic-responsive sigma(54)-dependent systems: a common phenotype by different mechanisms.
Open this publication in new window or tab >>Integration of global regulation of two aromatic-responsive sigma(54)-dependent systems: a common phenotype by different mechanisms.
2002 (English)In: Journal of Bacteriology, ISSN 0021-9193, E-ISSN 1098-5530, Vol. 184, no 3, 760-770 p.Article in journal (Refereed) Published
Abstract [en]

Pseudomonas-derived regulators DmpR and XylR are structurally and mechanistically related sigma(54)-dependent activators that control transcription of genes involved in catabolism of aromatic compounds. The binding of distinct sets of aromatic effectors to these regulatory proteins results in release of a repressive interdomain interaction and consequently allows the activators to promote transcription from their cognate target promoters. The DmpR-controlled Po promoter region and the XylR-controlled Pu promoter region are also similar, although homology is limited to three discrete DNA signatures for binding sigma(54) RNA polymerase, the integration host factor, and the regulator. These common properties allow cross-regulation of Pu and Po by DmpR and XylR in response to appropriate aromatic effectors. In vivo, transcription of both the DmpR/Po and XylR/Pu regulatory circuits is subject to dominant global regulation, which results in repression of transcription during growth in rich media. Here, we comparatively assess the contribution of (p)ppGpp, the FtsH protease, and a component of an alternative phosphoenolpyruvate-sugar phosphotransferase system, which have been independently implicated in mediating this level of regulation. Further, by exploiting the cross-regulatory abilities of these two circuits, we identify the target component(s) that are intercepted in each case. The results show that (i) contrary to previous speculation, FtsH is not universally required for transcription of sigma(54)-dependent systems; (ii) the two factors found to impact the XylR/Pu regulatory circuit do not intercept the DmpR/Po circuit; and (iii) (p)ppGpp impacts the DmpR/Po system to a greater extent than the XylR/Pu system in both the native Pseudomonas putida and a heterologous Escherichia coli host. The data demonstrate that, despite the similarities of the specific regulatory circuits, the host global regulatory network latches onto and dominates over these specific circuits by exploiting their different properties. The mechanistic implications of how each of the host factors exerts its action are discussed.

Identifiers
urn:nbn:se:umu:diva-26208 (URN)10.1128/JB.184.3.760-770.2002 (DOI)11790746 (PubMedID)
Available from: 2009-09-30 Created: 2009-09-30 Last updated: 2017-05-24
2. The role of the alarmone (p)ppGpp in sigma N competition for core RNA polymerase.
Open this publication in new window or tab >>The role of the alarmone (p)ppGpp in sigma N competition for core RNA polymerase.
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2003 (English)In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, 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.

Identifiers
urn:nbn:se:umu:diva-26210 (URN)10.1074/jbc.M209268200 (DOI)12421818 (PubMedID)
Available from: 2009-09-30 Created: 2009-09-30 Last updated: 2017-05-24
3. The guanosine tetraphosphate (ppGpp) alarmone, DksA and promoter affinity for RNA polymerase in regulation of σ54-dependent transcription
Open this publication in new window or tab >>The guanosine tetraphosphate (ppGpp) alarmone, DksA and promoter affinity for RNA polymerase in regulation of σ54-dependent transcription
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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.

Keyword
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:nbn:se:umu:diva-16715 (URN)10.1111/j.1365-2958.2006.05129.x (DOI)16629675 (PubMedID)
Available from: 2009-04-15 Created: 2009-04-15 Last updated: 2017-05-24Bibliographically approved
4. Dual by-pass mechanisms of RNA polymerase mutants: implications for ppGpp- and DksA-mediated control of σ54-dependent transcription
Open this publication in new window or tab >>Dual by-pass mechanisms of RNA polymerase mutants: implications for ppGpp- and DksA-mediated control of σ54-dependent transcription
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(English)Manuscript (preprint) (Other academic)
National Category
Cell and Molecular Biology
Identifiers
urn:nbn:se:umu:diva-5551 (URN)
Available from: 2006-11-16 Created: 2006-11-16 Last updated: 2017-05-24Bibliographically approved
5. The role of the alarmone ppGpp and DksA in regulation of σ54-dependent transcription in Pseudomonas putida
Open this publication in new window or tab >>The role of the alarmone ppGpp and DksA in regulation of σ54-dependent transcription in Pseudomonas putida
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(English)Manuscript (Other academic)
Identifiers
urn:nbn:se:umu:diva-5552 (URN)
Available from: 2006-11-16 Created: 2006-11-16 Last updated: 2017-05-24Bibliographically approved

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