COOPERATIVE AND ANTICOOPERATIVE EFFECTS IN BINDING OF THE FIRST AND 2ND PLASMID O-SYM OPERATORS TO A LACL TETRAMER - EVIDENCE FOR CONTRIBUTIONS OF NON-OPERATOR DNA-BINDING BY WRAPPING AND LOOPING

The interaction of Inc operator DNA with inc repressor (LacI) is a cla ssic example of a genetic regulatory switch. To dissect the role of st oichiometry, subunit association, and effects of DNA length in positio ning this switch, we have determined binding isotherms for the interac tion of LacI with a high affinity (O-sym) operator on linearized plasm id (2500 bp) DNA over a wide range of macromolecular concentrations (1 0(-14) to 10(-8) M). Binding data were analyzed using a thermodynamic model involving four equilibria: dissociation of tetramers (T) into di mers (D), and binding of operator-containing plasmid DNA (O) to dimers and tetramers to form three distinct complexes, DO, TO, and TO2. Over the range of concentrations of repressor, operator, and salt (0.075 M K+ to 0.40 M K+) investigated, we find no evidence for any significan t thermodynamic effect of LacI dimers. Instead, all isotherms can be i nterpreted in terms of just two equilibria, involving only T and the T O and TO2 complexes. As a reference binding equilibrium, which we prop ose must approximate the DO binding interaction, we compare the plasmi d O-sym results with our extensive studies of the binding of a 40 bp O -sym DNA fragment to LacI. On this basis, we obtain a lower bound on t he LacI dimer-tetramer equilibrium constant and values of the equilibr ium constants for formation of TO and TO2 complexes. At a salt concent ration of 0.40 M, the O-sym plasmid binding data are consistent with a model with two independent and identical binding sites for operator p er LacI tetramer, in which the binding to a site on the tetramer is on ly slightly more favorable than the reference binding interaction. Inc reasingly large deviations from the independent-site model are observe d as the salt concentration is reduced; binding of a second operator t o form TO2 becomes strongly disfavored relative to formation of TO at low salt concentrations (0.075 to 0.125 M). In addition, binding of bo th the first and second plasmid operator DNA molecules to the tetramer becomes increasingly more favorable than the reference binding intera ction as [K+] is reduced from 0.40 M to 0.125 M. At 0.075 M K+, howeve r, the strength of binding of the second plasmid operator DNA to the L acI tetramer is dramatically reduced; this interaction is much less fa vorable than binding the first plasmid operator DNA, and becomes much less favorable than the reference binding interaction. We propose that these differences arise from changes in the nature of the TO and TO2 complexes with decreasing salt concentration. At low salt concentratio n, we suggest the hypothesis that flanking non-operator sequences bind non-specifically (coulombically) by local wrapping, and that distant regions of non-operator DNA occupy the second operator-binding site by looping. We propose that wrapping stabilizes both 1:1 and 2:1 complex es at low salt concentration, and that looping stabilizes the 1:1 comp lex but competitively destabilizes the 2:1 TO2 complex at low salt con centration. These effects must play a role in adjusting the stability and structure of the LacI-lac operator repression complex as the cytop lasmic [K+] varies in response to changes in extracellular osmolarity.