TY - JOUR
T1 - Mean DNA bend angle and distribution of DNA bend angles in the CAP-DNA complex in solution
AU - Kapanidis, Achillefs N.
AU - Ebright, Yon W.
AU - Ludescher, Richard D.
AU - Chan, Shirley
AU - Ebright, Richard H.
N1 - Funding Information:
We thank Y. Ma for development of procedures for site-specific incorporation of AEDANS into CAP; R. Austin for preliminary experiments and discussions; Y. Jia and R. Hochstrasser for access to the Regional Laser and Biotechnology Laboratory, Philadelphia, PA, where pilot experiments were performed; and J. Beechem, H. Berman, S. Chen, E. Gratton, W. Olson, and A. Srinivasan for discussions. A.N.K. was partially supported by the State Scholarship Foundation, Athens, Greece. This work was supported by NIH grant GM41376 and a Howard Hughes Medical Institute Investigatorship to R.H.E.
PY - 2001/9/21
Y1 - 2001/9/21
N2 - 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)°). Lifetime-distribution analysis indicates that the distribution of DNA bend angles is relatively narrow, with <10% of DNA bend angles exceeding 100°. Millisecond time-resolved luminescence measurements using lanthanide-chelate probes provide independent evidence that the upper limit of the distribution of DNA bend angles is ∼100°. The methods used here will permit mutational analysis of CAP-induced DNA bending and the role of CAP-induced DNA bending in transcriptional activation.
AB - 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)°). Lifetime-distribution analysis indicates that the distribution of DNA bend angles is relatively narrow, with <10% of DNA bend angles exceeding 100°. Millisecond time-resolved luminescence measurements using lanthanide-chelate probes provide independent evidence that the upper limit of the distribution of DNA bend angles is ∼100°. The methods used here will permit mutational analysis of CAP-induced DNA bending and the role of CAP-induced DNA bending in transcriptional activation.
KW - Catabolite activator protein (CAP)
KW - Fluorescence resonance energy transfer
KW - Protein-induced DNA bending
KW - Transcriptional activation
KW - cAMP receptor protein (CRP)
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U2 - 10.1006/jmbi.2001.4976
DO - 10.1006/jmbi.2001.4976
M3 - Article
C2 - 11563909
AN - SCOPUS:0035929295
SN - 0022-2836
VL - 312
SP - 453
EP - 468
JO - Journal of Molecular Biology
JF - Journal of Molecular Biology
IS - 3
ER -