High resolution microanalysis and three-dimensional nucleosome structure associated with transcribing chromatin

Abstract

The nucleosome is the ubiquitous and fundamental DNA-protein complex of the eukaryotic chromosome, participating in the packaging of DNA and in the regulation of gene expression. Biophysical studies have implicated changes in nucleosome structure from chromatin that is quiescent to active in transcription. Since DNA within the nucleosome contains a high concentration of phosphorus whereas histone proteins do not, the nucleosome structure is amenable to microanalytical electron energy loss mapping of phosphorus to delineate the DNA within the protein-nucleic acid particle. Nucleosomes associated with transcriptionally active genes were separated from nucleosomes associated with quiescent genes using mercury-affinity chromatography. The three-dimensional image reconstruction methods for the total nucleosome structure and for the 3D DNA-phosphorus distribution combined quaternion-assisted angular reconstitution of sets of single particles at random orientations and electron spectroscopic imaging. The structure of the active nucleosome has the conformation of an open clam-shell, C- or U-shaped in one view, elongated in another, and exhibits a protein asymmetry. A three-dimensional phosphorus map reveals a conformational change in nucleosomal DNA compared to DNA in the canonical nucleosome structure. It indicates an altered superhelicity and is consistent with unfolding of the particle. The results address conformational changes of the nucleosome and provide a direct structural linkage to biochemical and physiological changes which parallel gene expression.