Wrapping of flanking non-operator DNA in lac repressor-operator complexes: implications for DNA looping

Area of Science:

• Molecular Biology
• Biophysics
• Structural Biology

Background:

• Lac repressor (LacI) tetramer-lac operator (O) interactions are crucial for gene regulation.
• Previous studies noted unusual cooperative and anticooperative effects with flanking DNA.
• The influence of flanking DNA length and salt concentration on binding was not fully understood.

Purpose of the Study:

• To investigate the origin of unusual binding effects of flanking DNA on lac repressor.
• To analyze the impact of varying flanking DNA lengths (40 bp, 101 bp, 2514 bp) on repressor-operator complex formation.
• To quantify the contributions of DNA wrapping modes to binding thermodynamics across a wide salt concentration range.

Main Methods:

• Binding assays were performed to measure repressor-operator complex formation (1:1 and 1:2 complexes).
• Experiments were conducted with a single symmetric lac operator (O(sym)) embedded in DNA fragments of different lengths.
• Binding constants (Kobs) and their dependence on salt concentration were analyzed to determine free energy contributions.

Main Results:

• Flanking DNA length and salt concentration significantly and interactively affect binding constants.
• Two DNA wrapping modes, 'local' and 'global', were identified, increasing with decreasing salt concentration.
• Global wrapping of long DNA is highly salt-dependent, driven by polyelectrolyte effects and involving extensive electrostatic interactions.

Conclusions:

• DNA wrapping, particularly global wrapping of long DNA, plays a critical role in lac repressor-operator binding.
• Global wrapping involves significant electrostatic interactions and unfavorable non-coulombic contributions, suggesting DNA distortion and entropic constraints.
• DNA wrapping may be a general mechanism employed by DNA-binding proteins to organize flanking DNA.