title: DNA-binding properties of a lac repressor mutant incapable of forming tetramers.
abstract: 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 (LacIadi) 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 repressor (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." LacIadi 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 Iadi mutation disrupts the monomer-dimer as well as eliminating the dimer-tetramer association equilibria while the DNA binding affinity of LacIadi 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 LacIadi and Gal repressor, a protein believed to function by mediating the formation of a looped complex, are discussed.
authors.affiliation: Department of Biochemistry, Albert Einstein College of Medicine, Bronx, New York 10461.
authors.forename: M
authors.initials: M
authors.lastname: Brenowitz
authors.forename: N
authors.initials: N
authors.lastname: Mandal
authors.forename: A
authors.initials: A
authors.lastname: Pickar
authors.forename: E
authors.initials: E
authors.lastname: Jamison
authors.forename: S
authors.initials: S
authors.lastname: Adhya
mesh_terms: D001345:Autoradiography; D001665:Binding Sites; D002850:Chromatography, Gel; D004269:DNA, Bacterial / Q000378:metabolism*; D009154:Mutation; D010957:Plasmids; D012097:Repressor Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*
chemical_list: D004269:DNA, Bacterial; D012097:Repressor Proteins