Probing the structure of gal operator-repressor complexes. Conformation change in DNA.
The gal operon is regulated by binding of Gal repressor to two operator loci, OE and OI, which are separated by 114 base pairs (bp). We have probed the actual operator DNA segments with and without Gal repressor occupation by characterizing the regions protected by repressor from DNase I digestion and dimethyl sulfate methylation. The segments which are protected from DNase I digestion in both OE and OI are about 22 bp long and seem to include 2-3 extra bp on either side of a 16-bp similar sequence containing an approximate dyad symmetry, with a consensus half-symmetry sequence GTG(G/T)AA-C. Repressor occupation hinders the reactivity of the consensus guanines in the four half-symmetry sequences, as shown by retardation of methylation at the N-7 positions by dimethyl sulfate owing to repressor binding. The protected guanines are symmetrically located. Since a dimeric Gal repressor affects symmetrically located bases, it is consistent with the notion that each half-operator is occupied by a repressor subunit. Because the N-7 positions of methylation of guanines lie in the major grooves and the protected guanines are located at positions 1, 3, 8 and the rotational 1', 3', and 8' in the 16-bp dyad symmetry, we suggest that Gal repressor establishes direct contacts with bases at 1, 3, 1', and 3' through two major grooves lying on one face of an operator helix and prevents reactivity of the guanines at 8 and 8' of a third major groove on the opposite face by changing the DNA helical structure at this position. Contacts at other positions are also discussed.
Majumdar
A
A
Laboratory of Molecular Biology, National Cancer Institute, Bethesda, Maryland 20892.
Adhya
S
S
D001483:Base Sequence; D004269:DNA, Bacterial / Q000235:genetics* / Q000648:ultrastructure; D004926:Escherichia coli / Q000235:genetics*; D005690:Galactose / Q000096:biosynthesis*; D006860:Hydrogen Bonding; D009690:Nucleic Acid Conformation; D009876:Operon; D011401:Promoter Regions, Genetic; D012097:Repressor Proteins / Q000378:metabolism*; D014157:Transcription Factors / Q000378:metabolism*
D004269:DNA, Bacterial; D012097:Repressor Proteins; D014157:Transcription Factors; D005690:Galactose