The H-NS protein represses transcription of the eltAB operon, which encodes heat-labile enterotoxin in enterotoxigenic Escherichia coli, by binding to regions downstream of the promoter.
Heat-labile enterotoxin, a major virulence determinant of enterotoxigenic Escherichia coli, is encoded by the eltAB operon. To elucidate the molecular mechanism by which the heat-stable nucleoid-structural (H-NS) protein controls transcription of eltAB, the authors constructed an eltAB-lacZ transcriptional fusion and performed beta-galactosidase analysis. The results showed that H-NS protein exerts fivefold repression on transcription from the eltAB promoter at 37 degrees C and 10-fold repression at 22 degrees C. Two silencer regions that were required for H-NS-mediated repression of eltAB expression were identified, both of which were located downstream of the start site of transcription. One silencer was located between +31 and +110, the other between +460 and +556, relative to the start site of transcription, and they worked cooperatively in repression. DNA sequences containing the silencers were predicted to be curved by in silico analysis and bound H-NS protein directly in vitro. Repression of eltAB transcription by H-NS was independent of promoter strength, and the presence of H-NS protein did not affect promoter opening in vitro, indicating that repression was achieved by inhibiting promoter clearance or blocking transcription elongation, probably via DNA looping between the two silencers.
Yang
Ji
J
Department of Microbiology and Immunology, University of Melbourne, Victoria 3010, Australia, and Murdoch Childrens Research Institute, Royal Children's Hospital, Flemington Road, Parkville, Victoria 3052, Australia.
Tauschek
Marija
M
Department of Microbiology and Immunology, University of Melbourne, Victoria 3010, Australia, and Murdoch Childrens Research Institute, Royal Children's Hospital, Flemington Road, Parkville, Victoria 3052, Australia.
Strugnell
Richard
R
Department of Microbiology and Immunology, University of Melbourne, Victoria 3010, Australia, and Murdoch Childrens Research Institute, Royal Children's Hospital, Flemington Road, Parkville, Victoria 3052, Australia.
Robins-Browne
Roy M
RM
Department of Microbiology and Immunology, University of Melbourne, Victoria 3010, Australia, and Murdoch Childrens Research Institute, Royal Children's Hospital, Flemington Road, Parkville, Victoria 3052, Australia.
D019677:Artificial Gene Fusion; D001426:Bacterial Proteins / Q000378:metabolism*; D001427:Bacterial Toxins / Q000235:genetics*; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D004269:DNA, Bacterial / Q000235:genetics / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004768:Enterotoxins / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism* / Q000472:pathogenicity; D029968:Escherichia coli Proteins / Q000235:genetics* / Q000378:metabolism*; D020868:Gene Silencing; D005798:Genes, Bacterial; D007763:Lac Operon; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009876:Operon; D011401:Promoter Regions, Genetic; D012097:Repressor Proteins / Q000378:metabolism*; D017384:Sequence Deletion; D014158:Transcription, Genetic
D001426:Bacterial Proteins; D001427:Bacterial Toxins; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; D004768:Enterotoxins; D029968:Escherichia coli Proteins; C031908:H-NS protein, bacteria; D012097:Repressor Proteins; C027538:heat-labile enterotoxin, E coli