Molecular flexibility of extended and compacted polynucleosomes. A steady-state fluorescence polarization study

We have studied the effects of Na+ (5-120 mM) and Mg2+ (0-6 mM) on the internal and overall flexibility of polynucleosome fragments from nuclease-solubilized chromatin from Ehrlich ascites cells. The mobility was monitored by the steady-state fluorescence polarization of the intercalated ethidium cation. The internal polynucleosome flexibility decreases continuously as the extended chromatin fragments are being compacted at increasing salt concentrations, and it can be further suppressed at ionic strengths above those where the 30 nm fiber is formed. The effect may be visualized as an initial formation of a loose 30 nm fiber that is further compacted at increasing ionic strengths. We observe several differences in the effects of Na+ and Mg2+ upon chromatin compaction. First, chromatin compacted by Mg2+ is less flexible than that compacted by Na+, suggesting a "tighter" chromatin structure with Mg2+. Second, Mg2+ affects the internal mobility in polynucleosome fragments shorter than 6-7 nucleosomes, which are too short to be compacted with Na+. Third, Mg2+ causes extensive macroscopic aggregation at concentrations above 0.2-0.3 mM, but the aggregation is uncorrelated with the intramolecular compaction. A quantitative evaluation of the overall polynucleosome "tumbling" mobility indicates that the compacted fragments possess more internal flexibility than do corresponding high molecular weight chromatin fibers. Finally, we note a correlation between the ethidium binding constant and the internal chromatin flexibility, possibly arising from lower torsional and "unwinding" flexibility of the linker DNA segments of compacted chromatin fibers.