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

In order to define the mean DNA bend angle and distribution of DNA bend ang les 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 betwee n 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 t hat the mean DNA bend angle is 77(+/-3)' - consistent with the mean DNA ben d angle observed in crystallographic structures (80(+/- 12)degrees). Lifeti me-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-chela te probes provide independent evidence that the upper limit of the distribu tion of DNA bend angles is similar to 100 degrees. The methods used here wi ll permit mutational analysis of CAP-induced DNA bending and the role of CA P-induced DNA bending in transcriptional activation. (C) 2001 Academic Pres s.