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Protein-DNA interaction investigated by binding Escherichia coli lac repressor protein to poly[d(A · U-HgX)]

1976; Elsevier BV; Volume: 103; Issue: 1 Linguagem: Inglês

10.1016/0022-2836(76)90050-4

1089-8638

Timothy J. Richmond, Thomas A. Steitz,

Bacteriophages and microbial interactions

From studies of repressor binding to poly[d(A · U-HgX)], a structural analog of poly[d(A · T)], we demonstrate that the lac repressor does not make extensive contacts in the major groove of DNA or denature the DNA double helix in order to have a high affinity for non-operator DNA. Poly[d(A · U-HgX)] differs from poly-[d(A · T)] at the five position of the pyrimidine bases where a covalently bound mercury atom and associated mercaptan, X, replace the methyl group of thymidine. By selection of the appropriate mercaptan, the major groove of the DNA has been modified sterically and ionically at each base-pair. Space-filling models of these DNA derivatives show that the major groove is almost fully packed by mercaptans and that the two strands can be crosslinked by dithiothreitol. That dithiothreitol prevents this mercuriDNA from undergoing a normal thermal melting transition, provides evidence that this dithiol is cross-linking the two DNA chains. These modified DNAs bind well to the repressor (Kdis = 10−7 to 10−9 m). Thus, the high affinity of repressor for these non-operator DNA molecules cannot involve denaturation of the DNA duplex or binding contacts in the major groove. These studies make those models for repressor-DNA interaction which involve binding of an α-helix in the major groove of the DNA or binding of the repressor to cruciform or Gierer-like structures (1966) not possible for non-operator DNA. We conclude that repressor-operator interaction in the minor groove and/or sequence-dependent alteration of the double-stranded DNA conformation are probably important in repressor specificity.