Mapping the transition state for DNA bending by IHF

Area of Science:

• Molecular Biology
• Biophysics
• Structural Biology

Background:

• Understanding how DNA-binding proteins recognize specific DNA sequences is crucial.
• Indirect readout mechanisms rely on DNA sequence-dependent flexibility.
• Distinguishing between protein-induced DNA bending and conformational capture is challenging.

Purpose of the Study:

• To elucidate the DNA recognition mechanism of Escherichia coli integration host factor (IHF).
• To investigate the role of DNA flexibility and prebent conformations in IHF binding.
• To capture transition state snapshots of the DNA-protein recognition pathway.

Main Methods:

• Utilized fluorescence resonance energy transfer (FRET) measurements.
• Employed laser temperature-jump perturbation to monitor DNA bending dynamics.
• Analyzed DNA bending rates and binding affinities under various conditions.

Main Results:

• Enhanced DNA flexibility at kink sites increased IHF bending rates (3-4 fold) and binding affinities (4-11 fold).
• Modifications away from kink sites or IHF mutations had minimal effect on bending rates.
• These changes occurred despite significant decreases in binding affinities (>250 fold) for modified IHF.

Conclusions:

• The primary recognition bottleneck for IHF involves spontaneous DNA kinking into a prebent conformation.
• Conformational capture is the dominant mechanism for initial IHF-DNA recognition.
• Further protein-induced bending occurs after the transition state is reached.