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

• Published in: The Journal of biological chemistry Vol. 266; no. 2; pp. 1281 - 1288
• Main Authors: Brenowitz, M, Mandal, N, Pickar, A, Jamison, E, Adhya, S
• Format: Journal Article
• Language: English
• Published: Bethesda, MD Elsevier Inc 15-01-1991; American Society for Biochemistry and Molecular Biology
• Subjects: Autoradiography; Binding Sites; Biological and medical sciences; Chromatography, Gel; DNA, Bacterial - metabolism; Fundamental and applied biological sciences. Psychology; Interactions. Associations; Intermolecular phenomena; Molecular biophysics; Mutation; Plasmids; Repressor Proteins - genetics; Repressor Proteins - isolation & purification; Repressor Proteins - metabolism; Plasmid; Molecular cooperativity; Regulation(control); Transcription; Operon; DNA binding protein; Footprinting technique; Quantitative analysis; Conformation
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/S0021-9258(17)35313-9

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.