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

Journal article

In order to define the mean DNA bend angle and distribution of DNA bend angles in the catabolite activator protein (CAP)-DNA complex in solution under standard transcription initiation conditions, we have performed nanosecond time-resolved fluorescence measurements quantifying energy transfer between a probe incorporated at a specific site in CAP, and a complementary probe incorporated at each of five specific sites in DNA. The results indicate that the mean DNA bend angle is 77(+/-3) degrees - consistent with the mean DNA bend angle observed in crystallographic structures (80(+/-12) degrees ). Lifetime-distribution analysis indicates that the distribution of DNA bend angles is relatively narrow, with <10 % of DNA bend angles exceeding 100 degrees. Millisecond time-resolved luminescence measurements using lanthanide-chelate probes provide independent evidence that the upper limit of the distribution of DNA bend angles is approximately 100 degrees. The methods used here will permit mutational analysis of CAP-induced DNA bending and the role of CAP-induced DNA bending in transcriptional activation.

Authors: Kapanidis, A; Ebright, Y; Ludescher, R; Chan, S; Ebright, R

• Journal: Journal of molecular biology
• Volume: 312
• Issue: 3
• Pages: 453-468
• Publication date: 2001-09-01
• DOI: 10.1006/jmbi.2001.4976
• EISSN: 1089-8638
• ISSN: 0022-2836
• Language: English
• Keywords: Buffers; Binding Sites; DNA; Solutions; Fluorescence; Cyclic AMP Receptor Protein; Nucleic Acid Conformation; Protein Conformation; Transcription, Genetic; Energy Transfer; Fluorescence Polarization; DNA-Binding Proteins; Protein Binding; Computer Simulation; Models, Molecular; Response Elements; Base Sequence