Formation of nucleosomes on positively supercoiled DNA.

Abstract

A transcribing RNA polymerase is thought to generate positive supercoils in front of the advancing transcription complex and negative supercoils behind. We have examined the possibility that positive supercoils might destabilize nucleosomes, facilitating transcription. We show that histone octamers bind to positively supercoiled DNA, and that after the complex is relaxed, 'classical' nucleosomes are present. We tested the possibility that nucleosomes on positively supercoiled DNA are in an altered (presumably more open) conformation, but revert to the classical structure only on release of this stress. However, circular dichroic spectra, and chemical cross-linking and modification of core histones, all suggest that the complexes initially formed on positively supercoiled DNA are classical nucleosomes. Although such structures are stable, their formation requires the plasmid to become more positively supercoiled, resulting in greater superhelical stress. In contrast, formation of nucleosomes on negatively supercoiled DNA relieves superhelical stress. In an exchange experiment in which equilibrium is achieved, nucleosomes transfer from positively to negatively supercoiled DNA, as predicted from the super-coiling free energies of the reactions. This suggests a mechanism for transcription of a gene assembled into chromatin, in which octamers are sequentially transferred from the region in front of the polymerase to the region behind.