Coupled energetics of λ cro repressor self-assembly and site-specific DNA operator binding II: cooperative interactions of cro dimers

• Published in: Journal of molecular biology 2000-09, Vol.302 (3), p.625-638
• Main Authors: Darling, Paul J., Holt, Jo M., Ackers, Gary K.
• Format: Article
• Language: English
• Subjects: Allosteric Site; Bacteriophage lambda - chemistry; bacteriophage λ; cooperativity; Cro protein; cro repressor; Deoxyribonuclease I - metabolism; Dimerization; DNA Footprinting; DNA, Bacterial - genetics; DNA, Bacterial - metabolism; DNA-Binding Proteins - chemistry; DNA-Binding Proteins - metabolism; Gene Expression Regulation, Bacterial; genetic switch; Models, Genetic; Mutation; Operator Regions, Genetic - genetics; Phage ^l; Protein Binding; protein-DNA interactions; Repressor Proteins - chemistry; Repressor Proteins - metabolism; Reproducibility of Results; Substrate Specificity; Templates, Genetic; Thermodynamics; Viral Proteins - chemistry; Viral Proteins - metabolism; Viral Regulatory and Accessory Proteins
• ISSN: 0022-2836; 1089-8638
• DOI: 10.1006/jmbi.2000.4050

The bacteriophage λ relies on interactions of the cI and cro repressors which self assemble and bind the two operators (O R and O L) of the phage genome to control the lysogenic to lytic switch. While the self assembly and O R binding of cI have been investigated in detail, a more complete understanding of gene regulation by phage λ also requires detailed knowledge of the role of cro repressor as it dimerizes and binds at O R sites. Since dimerization and operator binding are coupled processes, a full elucidation of the regulatory energetics in this system requires that the equilibrium constants for dimerization and cooperative binding be determined. The dimerization constant for cro has been measured as a prelude to these binding studies. Here, the energetics of cro binding to O R are evaluated using quantitative DNaseI footprint titration techniques. Binding data for wild-type and modified O R site combinations have been simultaneously analyzed in concert with the dimerization energetics to obtain both the intrinsic and cooperative DNA binding energies for cro with the three O R sites. Binding of cro dimers is strongest to O R3, then O R1 and lastly, O R2. Adjacently bound repressors exhibit positive cooperativity ranging from −0.6 to −1.0 kcal/mol. Implications of these, newly resolved, energetics are discussed in the framework of a dynamic model for gene regulation. This characterization of the DNA-binding properties of cro repressor establishes the foundation on which the system can be explored for other, more complex, regulatory elements such as cI- cro cooperativity.