Fluorescence energy transfer analysis of DNA structures containing several bulges and their interaction with CAP

被引:27
作者
Stühmeier, F
Hillisch, A
Clegg, RM
Diekmann, S
机构
[1] Loomis Lab Phys, Dept Phys, Urbana, IL 61801 USA
[2] Loomis Lab Phys, Fluorescence Dynam Lab, Urbana, IL 61801 USA
[3] Pfizer Ltd, Cent Res, Sandwich CT13 9NJ, Kent, England
[4] Inst Mol Biotechnol eV, Mol Biol Abt, D-07745 Jena, Germany
关键词
fluorescence anisotropy; fluorescein; rhodamine; protein-DNA interaction; DNA-protein binding;
D O I
10.1006/jmbi.2000.4089
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
DNA molecules with three bulges separated by double-stranded helical sections of B-DNA were constructed to be used as substrates for DNA-protein binding assays. Fluorescence resonance energy transfer (FRET) between dye molecules attached to the 5'-ends of the DNA molecules is used to monitor the protein binding. The A, bulge, which consists of five unpaired adenine nucleotides, alters the direction of the helical axis by approximately 80 to 90 degrees at every bulge site. Computer molecular modeling facilitated a pre-selection of suitable helix lengths that bring the labeled ends of the three-bulge DNA molecules (60 to 70 base-pairs long) into close proximity. The FRET experiments verified that the labeled ends of the helices of these long molecules were indeed close. A series of FRET experiments was carried out with two A, and two A(7) bulge molecules. The relative positions of the bulges were varied along the central helical DNA sequence (between the bulges) in order to determine the relative angular juxtapositions of the outlying helical arms flanking the central helical region. The global structural features of the DNA molecules are manifested in the FRET data. The FRET experiments, especially those of the two-bulge series, could be interpreted remarkably well with molecular models based on the NMR structure of the A,bulge. These models assume that the DNA molecules do not undergo large torsional conformational fluctuations at the bulge sites. The magnitude of the FRET efficiency attests to a relatively rigid structure for many of the long 5'-end-labeled molecules. The changes in the FRET efficiency of three-bulge structures containing the specific binding sequence of the catabolite activator protein (CAP) demonstrated significant deformation of the DNA upon binding of CAP. No direct interaction of CAP with the dyes was observed. (C) 2000 Academic Press.
引用
收藏
页码:1081 / 1100
页数:20
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