VISUALIZATION AND ANALYSIS OF UNFOLDED NUCLEOSOMES ASSOCIATED WITH TRANSCRIBING CHROMATIN

We have characterized the structure of transcriptionally active nucleo some subunits using electron spectroscopic imaging, Individual nucleos omes were analyzed in terms of total mass, DNA and protein content, wh ile the ensemble of images of active nucleosomes was used to calculate a three-dimensional reconstruction. Transcriptionally active nucleoso mes were separated from inactive nucleosomes by mercury-affinity chrom atography thus making it possible to compare their structures, The chr omatographic results combined with electron spectroscopic imaging conf irm 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 determi ned using quaternion-assisted angular reconstitution methods unites an d resolves the different electron microscopic views of the particle an d is concordant with a sulphydryl-exposing disruption of the H3-H4 tet ramer.