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) 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.
Kapanidis
A N
AN
Howard Hughes Medical Institute, Waksman Institute and Department of Chemistry, Piscataway, NJ 08854, USA.
Ebright
Y W
YW
Ludescher
R D
RD
Chan
S
S
Ebright
R H
RH
D001483:Base Sequence; D001665:Binding Sites; D002021:Buffers; D003198:Computer Simulation; D002373:Cyclic AMP Receptor Protein / Q000737:chemistry* / Q000378:metabolism*; D004247:DNA / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism; D004735:Energy Transfer; D005453:Fluorescence; D005454:Fluorescence Polarization; D008958:Models, Molecular; D009690:Nucleic Acid Conformation; D011485:Protein Binding; D011487:Protein Conformation; D020218:Response Elements / Q000235:genetics; D012996:Solutions; D014158:Transcription, Genetic
D002021:Buffers; D002373:Cyclic AMP Receptor Protein; D004268:DNA-Binding Proteins; D012996:Solutions; D004247:DNA