Proton-linked contributions to site-specific interactions of lambda cI repressor and OR

Area of Science:

• Molecular Biology
• Biophysics
• Biochemistry

Background:

• The cI repressor protein regulates gene expression by binding to specific operator DNA sequences.
• Understanding the factors influencing repressor-operator binding is key to deciphering gene regulation mechanisms.

Purpose of the Study:

• To investigate the role of proton activity (pH) in the site-specific binding of cI repressors to operator sites.
• To determine the Gibbs energies of binding and cooperativity across a pH range (5-8).
• To reevaluate existing models of cooperativity in the cI repressor-operator system.

Main Methods:

• DNase I footprint titration was employed to study repressor-operator interactions.
• Individual-site binding isotherms were generated for wild-type and mutant operators.
• Gibbs energies and protonation effects (delta nu bar(H)) were calculated at various pH levels.

Main Results:

• Proton-linked effects significantly contribute to the differential affinities of cI repressor for the three operator sites.
• Acidic pH dramatically influences binding, suggesting roles for non-contacting repressor groups and conformational changes.
• Cooperative interaction between OR1 and OR3 was consistently absent, and data challenged the selection rule for OR2-OR3 cooperativity.

Conclusions:

• Site specificity in cI repressor-operator binding is influenced by ionizable repressor groups not directly contacting DNA and by conformational differences.
• The study provides a detailed analysis of protonation effects on protein-DNA interactions.
• Traditional models of cooperativity require reevaluation based on these findings.