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Identification of ligands affecting the activity of the transcriptional repressor CcpN from Bacillus subtilis.
J Mol Biol. 2008 Jun 27; 380(1):17-30.JM

Abstract

Carbon catabolite repression in Bacillus subtilis is mediated primarily by the major regulator CcpA. However, sugar-dependent repression of three genes, sr1 encoding a small nontranslated RNA and two genes coding for gluconeogenic enzymes, gapB and pckA, is carried out by the transcriptional repressor CcpN (control catabolite protein of gluconeogenic genes). It has previously been shown that ccpN is constitutively expressed, which leads to a constant occupation of all operators with CcpN. Since this would not allow for specific regulation, a ligand that modulates CcpN activity is required. In vitro transcription assays demonstrated that CcpN is able to specifically repress transcription to a small extent at the three mentioned promoters in the absence of an activating ligand. Upon testing of several ligands, including nucleotides and glycolysis intermediates, it could be shown that ATP is able to specifically enhance the repressing activity of CcpN, and this effect was more pronounced at a slightly acidic pH. Furthermore, ADP was found to specifically counteract the repressive effect of ATP. Circular dichroism measurements demonstrated a significant alteration of CcpN structure in the presence of ATP at acidic pH and in the presence of ADP. Electrophoretic mobility shift assays revealed that neither ATP nor ADP altered the affinity of CcpN for its operators. Therefore, we hypothesise that the effect of ligand-bound CcpN on the RNA polymerase might be due to a conformational switch that alters the interaction between the two proteins. Based on these results, a working model for CcpN action is discussed.

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Authors+Show Affiliations

Licht A
AG Bakteriengenetik, Friedrich-Schiller-Universität Jena, Philosophenweg 12, D-07743 Jena, Germany. andreas.licht@uni-jena.de
Golbik R
No affiliation info available
Brantl S
No affiliation info available

MeSH

Adenosine DiphosphateAdenosine TriphosphateBacillus subtilisBacterial ProteinsCarbonCircular DichroismDNA, BacterialDNA-Binding ProteinsElectrophoretic Mobility Shift AssayGene Expression Regulation, BacterialGenes, BacterialGlycolysisHydrogen-Ion ConcentrationLigandsModels, GeneticMutant ProteinsMutationPromoter Regions, GeneticProtein Structure, TertiaryRecombinant Fusion ProteinsRepressor ProteinsTranscription, Geneticbeta-Galactosidase

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't

Language

eng

PubMed ID

18511073

Citation

Licht, Andreas, et al. "Identification of Ligands Affecting the Activity of the Transcriptional Repressor CcpN From Bacillus Subtilis." Journal of Molecular Biology, vol. 380, no. 1, 2008, pp. 17-30.
Licht A, Golbik R, Brantl S. Identification of ligands affecting the activity of the transcriptional repressor CcpN from Bacillus subtilis. J Mol Biol. 2008;380(1):17-30.
Licht, A., Golbik, R., & Brantl, S. (2008). Identification of ligands affecting the activity of the transcriptional repressor CcpN from Bacillus subtilis. Journal of Molecular Biology, 380(1), 17-30. https://doi.org/10.1016/j.jmb.2008.05.002
Licht A, Golbik R, Brantl S. Identification of Ligands Affecting the Activity of the Transcriptional Repressor CcpN From Bacillus Subtilis. J Mol Biol. 2008 Jun 27;380(1):17-30. PubMed PMID: 18511073.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Identification of ligands affecting the activity of the transcriptional repressor CcpN from Bacillus subtilis. AU - Licht,Andreas, AU - Golbik,Ralph, AU - Brantl,Sabine, Y1 - 2008/05/08/ PY - 2008/02/07/received PY - 2008/04/29/revised PY - 2008/05/02/accepted PY - 2008/5/31/pubmed PY - 2008/6/26/medline PY - 2008/5/31/entrez SP - 17 EP - 30 JF - Journal of molecular biology JO - J Mol Biol VL - 380 IS - 1 N2 - Carbon catabolite repression in Bacillus subtilis is mediated primarily by the major regulator CcpA. However, sugar-dependent repression of three genes, sr1 encoding a small nontranslated RNA and two genes coding for gluconeogenic enzymes, gapB and pckA, is carried out by the transcriptional repressor CcpN (control catabolite protein of gluconeogenic genes). It has previously been shown that ccpN is constitutively expressed, which leads to a constant occupation of all operators with CcpN. Since this would not allow for specific regulation, a ligand that modulates CcpN activity is required. In vitro transcription assays demonstrated that CcpN is able to specifically repress transcription to a small extent at the three mentioned promoters in the absence of an activating ligand. Upon testing of several ligands, including nucleotides and glycolysis intermediates, it could be shown that ATP is able to specifically enhance the repressing activity of CcpN, and this effect was more pronounced at a slightly acidic pH. Furthermore, ADP was found to specifically counteract the repressive effect of ATP. Circular dichroism measurements demonstrated a significant alteration of CcpN structure in the presence of ATP at acidic pH and in the presence of ADP. Electrophoretic mobility shift assays revealed that neither ATP nor ADP altered the affinity of CcpN for its operators. Therefore, we hypothesise that the effect of ligand-bound CcpN on the RNA polymerase might be due to a conformational switch that alters the interaction between the two proteins. Based on these results, a working model for CcpN action is discussed. SN - 1089-8638 UR - https://www.unboundmedicine.com/medline/citation/18511073/Identification_of_ligands_affecting_the_activity_of_the_transcriptional_repressor_CcpN_from_Bacillus_subtilis_ L2 - https://linkinghub.elsevier.com/retrieve/pii/S0022-2836(08)00544-5 DB - PRIME DP - Unbound Medicine ER -