The chromatin-associated protein H-NS alters DNA topology in vitro.
H-NS is one of the two most abundant proteins in the bacterial nucleoid 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 constrain negative DNA supercoils in vitro. This provides support for the hypothesis that H-NS influences transcription via changes in DNA topology, and is evidence of a structural role for H-NS in bacterial chromatin. The effects of H-NS on topology were only observed at sub-saturating 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 element which we previously showed, by in vivo studies, to be a site at which H-NS influences transcription of the proU operon. When present in saturating concentrations, H-NS did not constrain supercoils and bound to DNA in a sequence-independent fashion, covering all DNA molecules from end to end, suggesting that H-NS may form distinct complexes with DNA at different H-NS:DNA ratios. The data presented here provide direct support for the hypothesis that H-NS acts at specific sites to influence DNA topology and, hence, transcription.
Tupper
A E
AE
Imperial Cancer Research Fund Laboratories, University of Oxford, John Radcliffe Hospital, UK.
Owen-Hughes
T A
TA
Ussery
D W
DW
Santos
D S
DS
Ferguson
D J
DJ
Sidebotham
J M
JM
Hinton
J C
JC
Higgins
C F
CF
D001426:Bacterial Proteins; D001483:Base Sequence; D002843:Chromatin / Q000378:metabolism*; D004269:DNA, Bacterial / Q000737:chemistry* / Q000378:metabolism / Q000648:ultrastructure; D004268:DNA-Binding Proteins / Q000378:metabolism* / Q000648:ultrastructure; D003850:Deoxyribonuclease I; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000378:metabolism* / Q000648:ultrastructure; D009690:Nucleic Acid Conformation; D009994:Osmolar Concentration; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D012486:Salmonella typhimurium; D014158:Transcription, Genetic
D001426:Bacterial Proteins; D002843:Chromatin; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; C031908:H-NS protein, bacteria; D009687:Nuclear Proteins; D003850:Deoxyribonuclease I