Thermodynamics of the interactions of Lac repressor with variants of the symmetric Lac operator: Effects of converting a consensus site to a non-specific site

Diane E. Frank, Ruth M. Saecker, Jeffrey P. Bond, Michael W. Capp, Oleg V. Tsodikov, Sonya E. Melcher, Mark M. Levandoski, M. Thomas Record

Research output: Contribution to journalArticlepeer-review

102 Scopus citations


What are the thermodynamic consequences of the stepwise conversion of a highly specific (consensus) protein-DNA interface to one that is non-specific? How do the magnitudes of key favorable contributions to complex stability (burial of hydrophobic surfaces and reduction of DNA phosphate charge density) change as the DNA sequence of the specific site is detuned? To address these questions we investigated the binding of lac repressor (LacI) to a series of 40 bp fragments carrying symmetric (consensus) and variant operator sequences over a range of temperatures and salt concentrations. Variant DNA sites contained symmetrical single and double base-pair substitutions at positions 4 and/or 5 in each 10 bp half site of the symmetric lac operator (O(sym)). Non-specific interactions were examined using a 40 bp non-operator DNA fragment. Disruption of the consensus interface by a single symmetrical substitution reduces the observed equilibrium association constant (K(obs)) for O(sym) by three to four orders of magnitude; double symmetrical substitutions approach the six orders in magnitude difference between specific and non-specific binding to a 40 bp fragment. At these adjacent positions in the consensus site, the free energy effects of multiple substitutions are non-additive: the first reduces [ΔG(obs)(o)] by 3 to 5 kcal mol-1, approximately halfway to the non-specific level, whereas the second is less deleterious, reducing [ΔG(obs)(o)] by less than 3 kcal mol-1. Variant-specific dependences of K(obs) on temperature and salt concentration characterize these LacI-operator interactions. In general, binding constants and standard free energies of binding both exhibit characteristic extrema near 290 K. As a consequence, both the enthalpic and entropic contributions to stability of O(sym) and variant complexes change from positive (i.e. entropy driven) at lower temperatures to negative (i.e. enthalpy driven) at higher temperatures, indicating that the heat capacity change upon binding, ΔC(obs)(o), is large and negative. In general, [ΔC(obs)(o)] decreases as the specificity and stability of the variant complex decreases. Stabilities of complexes of LacI with O(sym) and all variant operators are strongly [salt]-dependent. Binding constants for the variant complexes exhibit a power-dependence on [salt] that is larger in magnitude (i.e. more negative) than for O(sym), but no obvious trend relates changes in contributions from the polyelectrolyte effect and the observed reductions in stability (ΔΔG(obs)(o)). These variant-specific thermodynamic signatures provide novel insights into the consequences of converting a consensus interface to a less specific one; such insights are not obtained from comparisons at the level of ΔΔG(obs)(o). We propose that this variant-specific behavior arises from a strong effect of operator sequence on the extent of induced conformational changes in the protein (and possibly also in the DNA site) which accompany binding.

Original languageEnglish
Pages (from-to)1186-1206
Number of pages21
JournalJournal of Molecular Biology
Issue number5
StatePublished - Apr 18 1997

Bibliographical note

Funding Information:
The authors thank S. Ross and Dr J.-H. Ha for contributions to this project in its early stages, Dr C. Bingman for assistance with surface area calculations, and Sheila Aiello for help in preparation of the manuscript. This work was supported by NIH research grant GM 23467. D.E.F. and M.M.L. were supported in part as NIH Cell and Molecular Biology trainees and S.E.M. as a NIH Molecular Biophysics trainee.


  • Consensus
  • Recognition
  • Specificity
  • lac repressor

ASJC Scopus subject areas

  • Molecular Biology
  • Biophysics
  • Structural Biology


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