Gross D S, English K E, Collins K W, Lee S W
Department of Biochemistry and Molecular Biology, Louisiana State University Medical Center, Shreveport 71130.
J Mol Biol. 1990 Dec 5;216(3):611-31. doi: 10.1016/0022-2836(90)90387-2.
We describe here for the first time successful application of the hydroxyl radical technique for genomic footprinting. In combination with two complementary techniques, DNase I footprinting and dimethyl sulfate methylation protection, we have obtained a high-resolution map of the promoter region of the yeast HSP82 heat shock gene, which resides within a constitutive nuclease hypersensitive site. We find that irrespective of transcriptional state, basal or induced, only one of three putative heat shock elements, HSE1, and the TATA box are tightly bound by proteins, presumably heat shock factor (HSF) and TFIID, respectively. Whereas the HSE1-associated factor binds tightly within the major groove of DNA, as discerned by protection of guanine residues from methylation by dimethyl sulfate in intact cells, the TATA factor appears to bind principally to the sugar-phosphate backbone, as revealed by strong protection from hydroxyl radical cleavage in whole-cell lysates. In addition, while HSE1 is strongly footprinted by DNase I in lysates, the TATA box is only weakly footprinted. Strikingly, both elements are associated with marked distortion of the DNA double helix in chromatin. Protein binding to HSE1 appears to cause a non-B-conformation, on the basis of a local 12 base-pair periodicity of hydroxyl radical protection and the presence of multiple DNase I hyperreactive cleavages flanking HSE1, whose pattern changes following heat shock. Similarly, helix distortion is evident in the vicinity of the TATA box, since hydroxyl radical detects a lower strand-specific hypersensitive site at the dyad center of an adjacent polypurine tract. Finally, the absence of discernable modulation in the DNase I cleavage pattern argues against the presence of a specifically positioned nucleosome within the IISP82 promoter region.
我们在此首次描述了羟基自由基技术在基因组足迹分析中的成功应用。结合两种互补技术，即DNase I足迹分析和硫酸二甲酯甲基化保护技术，我们获得了酵母HSP82热休克基因启动子区域的高分辨率图谱，该基因位于一个组成型核酸酶超敏位点内。我们发现，无论转录状态是基础状态还是诱导状态，三个假定的热休克元件中只有一个，即HSE1，以及TATA框分别被蛋白质紧密结合，推测分别是热休克因子（HSF）和TFIID。通过完整细胞中鸟嘌呤残基免受硫酸二甲酯甲基化的保护可以看出，与HSE1相关的因子紧密结合在DNA的大沟内，而通过全细胞裂解物中羟基自由基切割的强烈保护表明，TATA因子似乎主要结合在糖-磷酸主链上。此外，虽然HSE1在裂解物中被DNase I强烈标记，但TATA框仅被弱标记。引人注目的是，这两个元件都与染色质中DNA双螺旋的明显扭曲有关。基于羟基自由基保护的局部12个碱基对周期性以及HSE1两侧多个DNase I高反应性切割的存在，蛋白质与HSE1的结合似乎导致了一种非B构象，其模式在热休克后发生变化。同样，在TATA框附近也明显存在螺旋扭曲，因为羟基自由基在相邻多聚嘌呤序列的二分中心检测到较低的链特异性超敏位点。最后，DNase I切割模式中没有可察觉的调节，这表明在IISP82启动子区域不存在特定定位的核小体。
