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We report the measurement of two specific protein to DNA distances in several conformational states of core nucleosomes by singlet-singlet energy transfer. A distance of 50-53 A separates each DNA terminus from cysteine-110 of chicken erythrocyte histone H3 in the native nucleosome. This cysteine residue must therefore be located very near the center of the nucleosome. The H3-DNA distance remained nearly constant in several unfolded forms of the core particles, as found in very low salt, in 0.6 M NaCl, and in high urea. Furthermore, it was shown that each DNA end lies within 32 A of cysteine-73 of Arbacia lixula sperm histone H4 in both the compact and the low-salt unfolded forms of the nucleosome. Because of the invariance of the two measured distances in the various conformational states of the nucleosome, we conclude that the cysteine-containing C-terminal segments of histones H3 and H4 maintain a very strong and close association with the terminal positions of the 146 base pair nucleosomal DNA. This binding may provide the primary interactions necessary for the folding of DNA into nucleosomes and for protection of 146 base pair nucleosomes from further nuclease digestion.

The effect of DNA length on the low salt unfolding transition of nucleosomes has been studied by the use of fluorescently labeled histones. Nucleosomes were formed by the reconstitution of bulk DNA fragments averaging 173 and 250 base pairs in length. These nucleosomes exhibited a conformational change in a transition centered at about 7 mM ionic strength, very different from that observed for the standard 145 bp nucleosomes (1-3mM). In addition, the conformational change of the 173 and 250 bp nucleosomes involves twice as many ions as that of the 145 bp nucleosomes.

SynopsisT h e kinetics of a conformational change which occurs in nucleosome core particles a t ahout 1 m M ionic strength have been studied by observing changes in the fluorescence of labeled histone H3. T h e unfolding reaction is intramolecular since no concentration dependence is ohserved. However, the kinetics are unexpectedly complicated and reveal evidence of a t least three relaxation times. It is possible to fit the kinetics observed under several conditions to a consistent four-state cyclic mechanism in which folded and unfolded forms can interconvert by two parallel pathways, each involving a distinct intermediate. While the data are not sufficient to establish this mechanism as a unique choice, they exclude many simpler possibilities. T h e cyclic mechanism is quite reasonable in view of what is currently known ahout the structures of the folded and unfolded forms.

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