Mean DNA bend angle and distribution of DNA bend angles in the CAP-DNA complex in solution

Area of Science:

• Molecular Biology
• Biophysics

Background:

• The catabolite activator protein (CAP) is a crucial transcription factor that regulates gene expression in bacteria.
• Understanding the precise DNA bending induced by CAP is essential for elucidating its mechanism of transcriptional activation.

Purpose of the Study:

• To determine the mean DNA bend angle and the distribution of DNA bend angles in the CAP-DNA complex in solution.
• To investigate the role of CAP-induced DNA bending in transcription initiation.

Main Methods:

• Nanosecond time-resolved fluorescence resonance energy transfer (TR-FRET) measurements were employed to quantify energy transfer between probes on CAP and DNA.
• Millisecond time-resolved luminescence measurements using lanthanide-chelate probes were used for independent validation.

Main Results:

• The mean DNA bend angle was determined to be 77 ± 3 degrees, closely matching crystallographic observations (80 ± 12 degrees).
• Lifetime-distribution analysis revealed a narrow distribution of DNA bend angles, with less than 10% exceeding 100 degrees.
• Luminescence measurements confirmed that the upper limit of DNA bend angles is approximately 100 degrees.

Conclusions:

• The study provides precise measurements of DNA bending by CAP in solution, supporting its role in transcriptional regulation.
• The developed methods allow for future mutational analyses of CAP-DNA interactions and their impact on transcription.