Bazett-Jones D P, Mendez E, Czarnota G J, Ottensmeyer F P, Allfrey V G
Department of Anatomy, University of Calgary, Canada.
Nucleic Acids Res. 1996 Jan 15;24(2):321-9. doi: 10.1093/nar/24.2.321.
We have characterized the structure of transcriptionally active nucleosome subunits using electron spectroscopic imaging. Individual nucleosomes were analyzed in terms of total mass, DNA and protein content, while the ensemble of images of active nucleosomes was used to calculate a three-dimensional reconstruction. Transcriptionally active nucleosomes were separated from inactive nucleosomes by mercury-affinity chromatography thus making it possible to compare their structures. The chromatographic results combined with electron spectroscopic imaging confirm that active nucleosomes unfold to form extended U-shaped particles. Phosphorus mapping indicated that the nucleosomal DNA also underwent a conformational change consistent with particle unfolding. The three-dimensional structure of the Hg-affinity purified nucleosomes determined using quaternion-assisted angular reconstitution methods unites and resolves the different electron microscopic views of the particle and is concordant with a sulphydryl-exposing disruption of the H3-H4 tetramer.
我们利用电子光谱成像技术对转录活性核小体亚基的结构进行了表征。从总质量、DNA和蛋白质含量方面分析了单个核小体，同时利用活性核小体的图像集合计算出三维重建结构。通过汞亲和色谱法将转录活性核小体与非活性核小体分离，从而能够比较它们的结构。色谱结果与电子光谱成像相结合证实，活性核小体展开形成伸展的U形颗粒。磷映射表明核小体DNA也经历了与颗粒展开一致的构象变化。使用四元数辅助角重构方法确定的汞亲和纯化核小体的三维结构统一并解析了该颗粒不同的电子显微镜视图，并且与H3-H4四聚体的巯基暴露破坏相一致。
