Lorenz M, Hillisch A, Goodman S D, Diekmann S
Department of Molecular Biology, Institute for Molecular Biotechnology, PO Box 100813, D-07708 Jena, Germany.
Nucleic Acids Res. 1999 Dec 1;27(23):4619-25. doi: 10.1093/nar/27.23.4619.
We have analyzed the structure of two related protein-DNA complexes consisting of integration host factor (IHF) bound to two different versions of the H' site of bacteriophage lambda. Both DNA substrates were 55 bp in length. While one was native duplex the other possessed a nick in one strand at a crucial position within the IHF consensus at the same position as in the reported crystal structure of the DNA-IHF complex. By labeling the 5'-ends of these DNA molecules with donor and acceptor fluorescent dyes, we were able to measure the distance between the dyes by fluorescence resonance energy transfer (FRET) and model DNA distortion. The FRET efficiency decreased from 0.49 +/- 0.01 (nicked DNA) to 0.37 +/- 0.01 (intact DNA) when the gap in the DNA strand was closed. The measured dye-to-dye distance of IHF in complex with nicked DNA was in agreement with the expected value from the crystal structure. Although we found that the two structures were distinguishable, the global shape induced by IHF was retained between the two DNA molecules. Furthermore, our FRET and modeling techniques have sufficiently high resolution to distinguish subtle changes in nucleoprotein complexes with biological relevance.
我们分析了两个相关的蛋白质-DNA复合物的结构，它们由整合宿主因子（IHF）与噬菌体λ的两个不同版本的H'位点结合而成。两种DNA底物长度均为55 bp。一种是天然双链，另一种在与DNA-IHF复合物报道的晶体结构相同位置的IHF共有序列关键位置的一条链上有一个切口。通过用供体和受体荧光染料标记这些DNA分子的5'末端，我们能够通过荧光共振能量转移（FRET）和模拟DNA扭曲来测量染料之间的距离。当DNA链上的缺口闭合时，FRET效率从0.49±0.01（带切口的DNA）降至0.37±0.01（完整DNA）。与带切口DNA形成复合物的IHF的染料间测量距离与晶体结构的预期值一致。尽管我们发现这两种结构是可区分的，但IHF诱导的整体形状在两个DNA分子之间得以保留。此外，我们的FRET和建模技术具有足够高的分辨率，能够区分具有生物学相关性的核蛋白复合物中的细微变化。
