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 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)' - 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 similar to 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. (C) 2001 Academic Press.