HIGH-RESOLUTION MICROANALYSIS AND 3-DIMENSIONAL NUCLEOSOME STRUCTURE ASSOCIATED WITH TRANSCRIBING CHROMATIN

The nucleosome is the ubiquitous and Fundamental DNA-protein complex o f the eukaryotic chromosome, participating in the packaging of DNA and in the regulation of gene expression. Biophysical studies have implic ated 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 l ass mapping of phosphorus to delineate the DNA within the protein-nucl eic acid particle. Nucleosomes associated with transcriptionally activ e genes were separated from nucleosomes associated with quiescent gene s using mercury-affinity chromatography. The three-dimensional image r econstruction methods for the total nucleosome structure and for the 3 D DNA-phosphorus distribution combined quaternion-assisted angular rec onstitution of sets of single particles at random orientations and ele ctron spectroscopic imaging. The structure of the active nucleosome ha s the conformation of an open clam-shell, C-or U-shaped in one view, e longated in another, and exhibits a protein asymmetry. A three-dimensi onal phosphorus map reveals a conformational change in nucleosomal DNA compared to DNA in the canonical nucleosome structure. It indicates a n altered superhelicity and is consistent with unfolding of the partic le. The results address conformational changes of the nucleosome and p rovide a direct structural linkage to biochemical and physiological ch anges which parallel gene expression. (C) 1997 Elsevier Science Ltd. A ll rights reserved.