DNA looping in cellular repression of transcription of the galactose operon.
Communication between distant DNA sites is a central feature of many DNA transactions. Negative regulation of the galactose (gal) operon of Escherichia coli requires repressor binding to two operator sites located on opposite sides of the promoter. The proposed mechanism for regulation involves binding of the repressor to both operator sites, followed by a protein-protein association that loops the intervening promoter DNA (double occupancy plus association). To assess these requirements in vivo, we have previously converted gal operator sites to lac and shown that both operator sites must be occupied by the homologous repressor protein (Lac or Gal) for negative regulation of the gal operon. We have now addressed more directly the need for protein-protein association by the use of the converted operator sites and a mutant Lac repressor defective in association of the DNA-binding dimers. We have compared the biological and biochemical activity of two Lac repressors: the wild-type (tetramer) I+ form, in which the DNA-binding dimer units are tightly associated; and the mutant Iadi repressor, in which the dimer units do not associate effectively. The I+ repressor is an efficient negative regulator of the gal operon in vivo, but the Iadi mutant is an ineffective repressor. Purified I+ repressor efficiently forms DNA loops between operator sites that we have visualized by electron microscopy; the Iadi repressor fails to form DNA loops, although the protein binds effectively to both operator sites. From the clear correlation between looping in vitro and repression in vivo, we conclude that regulation of the gal operon depends on the association of repressor proteins bound to the two operator sites.(ABSTRACT TRUNCATED AT 250 WORDS)
Laboratory of Molecular Biology, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892.
N
N
Mandal
W
W
Su
R
R
Haber
S
S
Adhya
H
H
Echols
D004269:DNA, Bacterial / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D005690:Galactose / Q000235:genetics*; D015964:Gene Expression Regulation, Bacterial; D007763:Lac Operon; D008854:Microscopy, Electron; D009154:Mutation; D009690:Nucleic Acid Conformation; D009875:Operator Regions, Genetic; D009876:Operon; D012097:Repressor Proteins / Q000378:metabolism*; D014157:Transcription Factors / Q000378:metabolism*; D014158:Transcription, Genetic
D004269:DNA, Bacterial; D012097:Repressor Proteins; D014157:Transcription Factors; D005690:Galactose