THE CHROMATIN-ASSOCIATED PROTEIN H-NS ALTERS DNA TOPOLOGY IN-VITRO

H-NS is one of the two most abundant proteins in the bacterial nucleoi d and influences the expression of a number of genes. We have studied the interaction of H-NS with DNA; purified H-NS was demonstrated to co nstrain negative DNA supercoils in vitro. This provides support for th e hypothesis that H-NS influences transcription via changes in DNA top ology, and is evidence of a structural role for H-NS in bacterial chro matin. The effects of H-NS on topology were only observed at sub-satur ating concentrations of the protein. In addition, a preferred binding site on DNA was identified by DNase I footprinting at sub-saturating H -NS concentrations. This site corresponded to a curved sequence elemen t which we previously showed, by in vivo studies, to be a site at whic h H-NS influences transcription of the proU operon. When present in sa turating concentrations, H-NS did not constrain supercoils and bound t o DNA in a sequence-independent fashion, covering all DNA molecules fr om end to end, suggesting that H-NS may form distinct complexes with D NA at different H-NS:DNA ratios. The data presented here provide direc t support for the hypothesis that H-NS acts at specific sites to influ ence DNA topology and, hence, transcription.