DNA binding is not sufficient for H-NS-mediated repression of proU expression.
H-NS is a major component of bacterial chromatin and influences the expression of many genes. H-NS has been shown to exhibit a binding preference for certain AT-rich curved DNA elements in vitro. In this study we have addressed the factors that determine the specificity of H-NS action in vitro and in vivo. In bandshift studies, H-NS showed a slight binding preference for all curved sequences tested whether GC-based or AT-based; the specific architecture of the curve also influenced H-NS binding. In filter retention assays little difference in affinity could be detected for any sequence tested, including the downstream regulatory element (DRE) a downstream curved DNA element required for H-NS to repress transcription of the Salmonella typhimurium proU operon in vivo. A Kd of 1-2 microM was estimated for binding of H-NS to each of these sequences. In vivo, the distance between the proU promoter and the DRE, their relative orientations on the face of the DNA helix, and translation of the DRE had no major effect on proU regulation. None of the synthetic curved sequences tested could functionally replace the DRE in vivo. These data show that differential binding to curved DNA cannot account for the specificity of H-NS action in vivo. Furthermore, binding of H-NS to DNA per se is insufficient to repress the proU promoter. Thus, the DRE does not simply act as an H-NS binding site but must have a more specific role in mediating H-NS regulation of proU transcription.
Jordi
B J
BJ
Nuffield Department of Clinical Biochemistry, and Imperial Cancer Research Fund Laboratories, Institute of Molecular Medicine, University of Oxford, John Radcliffe Hospital, Oxford OX3 9DU, United Kingdom.
Fielder
A E
AE
Burns
C M
CM
Hinton
J C
JC
Dover
N
N
Ussery
D W
DW
Higgins
C F
CF
D026905:Amino Acid Transport Systems; D001425:Bacterial Outer Membrane Proteins / Q000378:metabolism*; D001426:Bacterial Proteins / Q000096:biosynthesis* / Q000235:genetics; D001483:Base Sequence; D002352:Carrier Proteins / Q000096:biosynthesis* / Q000235:genetics; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000737:chemistry* / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D015964:Gene Expression Regulation, Bacterial; D017930:Genes, Reporter; D007763:Lac Operon; D008961:Models, Structural; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D010957:Plasmids; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D012486:Salmonella typhimurium / Q000235:genetics / Q000378:metabolism*; D001616:beta-Galactosidase / Q000096:biosynthesis
D026905:Amino Acid Transport Systems; D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins; D002352:Carrier Proteins; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; C031908:H-NS protein, bacteria; C060774:ProU protein, Bacteria; D011993:Recombinant Fusion Proteins; D001616:beta-Galactosidase