DNA-binding properties of a lac repressor mutant incapable of forming tetramers

The interaction of proteins bound to sites widely separated on the genome is a recurrent motif in both prokaryotic and eukaryotic regulatory systems. Lac repressor mediates the formation of "DNA loops" by the simultaneous interaction of a single protein tetramer with two DNA-binding sites. The DNA-binding properties of a Lac repressor mutant (LacI^adi) deficient in the association of protein dimers to tetramers was investigated. The results of quantitative footprint and gel mobility-shift titrations suggest that the wild-type Lac represser (LacI⁺) binds cooperatively to two operator sites separated by 11 helical turns on a linear DNA restriction fragment by the formation of a "looped complex." LacI^adi binds to this two-site operator non-cooperatively and without formation of a looped complex. These results demonstrate that the dimer-tetramer association of LacI⁺ is directly responsible for its cooperative binding and its ability to mediate formation of a looped complex. The I^adi mutation disrupts the monomer-dimer as well as eliminating the dimer-tetramer association equilibria while the DNA binding affinity of LacI^adi to a single site is unchanged relative to the wild-type protein. These results suggest that DNA binding and dimer-tetramer association are functionally unlinked. The similarity of the DNA-binding properties of LacI^adi and Gal repressor, a protein believed to function by mediating the formation of a looped complex, are discussed.