Novel aspects of the structure of the Escherichia coli nucleoid investigated by a rapid sedimentation assay

The Escherichia coli nucleoid is maintained in its folded highly condensed state by constraints which involve RNA and protein. We have developed a rapid sedimentation assay to determine the state of folding of the membrane-free nucleoid. An approximate measure of the stability of the nucleoids under various conditions can then be estimated by measuring the temperature at which the nucleoids unfold. Using ethidium and gamma irradiation (which removes the negative supercoiling of the native nucleoid) as probes, it can be shown that there are two types of constraint involved in the condensation of the nucleoid. One of these constraints is destablized by ethidium but stabilized by negative supercoiling; the second constraint is unaffected by both ethidium and negative supercoiling. Several models can be proposed: (i) a DNA∙RNA duplex, (ii) a double-strand DNA (dsDNA)∙RNA triplex, (iii) DNA-protein interactions, (iv) a topological knot with RNA, and (v) a DNA tetraplex. The topological knot model is not consistent with the data and many combinations of the others can be excluded. If RNA is involved in both constraints then RNA∙DNA duplexes and dsDNA∙RNA triplexes are involved in stabilizing the nucleoid.