title: A thermodynamic model of the cooperative interaction between the archaeal transcription factor Ss-LrpB and its tripartite operator DNA.
abstract: Ss-LrpB is a transcription factor of the archaeon Sulfolobus solfataricus that belongs to the leucine-responsive regulatory protein family. This protein binds to three distinct binding sites in the control region of its own gene, suggestive of autoregulation. Here, we present a detailed study of the thermodynamic and conformational rules that govern the interaction between Ss-LrpB and its tripartite operator DNA. Lane-per-lane partition analysis of macroscopic binding state populations in electrophoretic mobility shift assays, probing binding to full-length, truncated and mutated forms of the operator, allowed determination of equilibrium association constants and cooperativity parameters. The resulting thermodynamic model demonstrates that the Ss-LrpB-operator regulatory complex is formed with a significant positive cooperativity, which is mostly arising from dimer-dimer interactions between pairs of adjacent binding sites. There is a constraint on the spacing between these binding sites, with a preference for a cis-alignment on the DNA helix and with a 16-bp linker yielding maximal pairwise cooperativity. DNase I footprinting assays demonstrated that the extent of Ss-LrpB-induced DNA deformations depends on linker length. The knowledge of the thermodynamic principles underlying the Ss-LrpB-operator interaction, presented here, will contribute to unraveling of the cis-regulatory code of Ss-LrpB autoregulation.
authors.affiliation: Research group of Microbiology, Department of Bio-engineering Sciences, Vrije Universiteit Brussel, Pleinlaan 2, B-1050 Brussels, Belgium. espeeter@vub.ac.be
authors.forename: Eveline
authors.initials: E
authors.lastname: Peeters
authors.forename: Liesbeth
authors.initials: L
authors.lastname: van Oeffelen
authors.forename: Marc
authors.initials: M
authors.lastname: Nadal
authors.forename: Patrick
authors.initials: P
authors.lastname: Forterre
authors.forename: Daniel
authors.initials: D
authors.lastname: Charlier
mesh_terms: D019843:Archaeal Proteins / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites; D003001:Cloning, Molecular; D003198:Computer Simulation; D018983:DNA Footprinting; D019641:DNA, Archaeal / Q000235:genetics / Q000378:metabolism; D004269:DNA, Bacterial / Q000235:genetics / Q000378:metabolism*; D024202:Electrophoretic Mobility Shift Assay; D046911:Macromolecular Substances / Q000378:metabolism; D009690:Nucleic Acid Conformation; D009875:Operator Regions, Genetic; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D025941:Protein Interaction Mapping; D055503:Protein Multimerization; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D048229:Sulfolobus solfataricus / Q000235:genetics*; D013816:Thermodynamics; D014158:Transcription, Genetic
chemical_list: D019843:Archaeal Proteins; D019641:DNA, Archaeal; D004269:DNA, Bacterial; D046911:Macromolecular Substances; D011994:Recombinant Proteins