A thermodynamic model of the cooperative interaction between the archaeal transcription factor Ss-LrpB and its tripartite operator DNA

• Modeling of the Ss-LrpB-operator interaction by determining binding parameters.
• Densitometric analysis of binding states in electrophoretic mobility shift assays.
• Cooperativity arising from pairwise interactions strongly depends on linker length.
• Binding site configuration affects the protein-DNA complex conformation.

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.