title: NrpRII mediates contacts between NrpRI and general transcription factors in the archaeon Methanosarcina mazei Gö1.
abstract: We report here on the formation of a complex between the two NrpR homologs present in Methanosarcina mazei Gö1 and their binding properties to the nifH and glnK(1) promoters. Reciprocal co-chromatography demonstrated that NrpRI forms stable complexes with NrpRII (at an NrpRI : NrpRII molar ratio of ∼ 1 : 3), which are not affected by 2-oxoglutarate. Promoter-binding, analyses using DNA-affinity chromatography and electrophoretic gel mobility shift assays, verified that NrpRII is not able to bind to either the nifH promoter or the glnK(1) promoter except when in complex with NrpRI. Specific binding of NrpRI to the nifH and glnK(1) promoters was shown to be highly sensitive to 2-oxoglutarate, regardless of whether only NrpRI, or NrpRI in complex with NrpRII, bound to the promoter. Finally, strong interactions between NrpRII and the general transcription factors TATA-binding proteins (TBP) 1-3 and the general transcription factor TFIIB (TFB) were demonstrated, interactions which are also sensitive to 2-oxoglutarate. On the basis of these findings we propose the following: under nitrogen sufficiency NrpRII binds from solution to either the nifH promoter or the glnK(1) promoter by simultaneously contacting NrpRI and TBP plus TFB, resulting in full repression of transcription; whereas, under nitrogen limitation, increasing 2-oxoglutarate concentrations significantly decrease the binding of NrpRI to the operator as well as the binding of NrpRII to TBP and TFB, ultimately allowing recruitment of RNA polymerase to the promoter.
authors.affiliation: Institut für Allgemeine Mikrobiologie, Christian-Albrechts Universität zu Kiel, Kiel, Germany.
authors.forename: Katrin
authors.initials: K
authors.lastname: Weidenbach
authors.forename: Claudia
authors.initials: C
authors.lastname: Ehlers
authors.forename: Jutta
authors.initials: J
authors.lastname: Kock
authors.forename: Ruth A
authors.initials: RA
authors.lastname: Schmitz
mesh_terms: D019843:Archaeal Proteins / Q000235:genetics / Q000378:metabolism*; D001667:Binding, Competitive; D015153:Blotting, Western; D024202:Electrophoretic Mobility Shift Assay; D019848:Gene Expression Regulation, Archaeal / Q000187:drug effects; D007656:Ketoglutaric Acids / Q000494:pharmacology; D017020:Methanosarcina / Q000235:genetics / Q000378:metabolism*; D008957:Models, Genetic; D009584:Nitrogen / Q000494:pharmacology; D010088:Oxidoreductases / Q000235:genetics; D010957:Plasmids / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000235:genetics*; D011485:Protein Binding / Q000187:drug effects; D014176:Protein Biosynthesis / Q000187:drug effects; D020133:Reverse Transcriptase Polymerase Chain Reaction; D035181:TATA-Box Binding Protein / Q000235:genetics / Q000378:metabolism; D035581:Transcription Factor TFIIB / Q000235:genetics / Q000378:metabolism; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*
chemical_list: D019843:Archaeal Proteins; C449645:GlnK1 protein, Methanosarcina mazei; D007656:Ketoglutaric Acids; D035181:TATA-Box Binding Protein; D035581:Transcription Factor TFIIB; D014157:Transcription Factors; D010088:Oxidoreductases; C029130:nitrogenase reductase; D009584:Nitrogen