Thermodynamic and alkylation interference analysis of the lac repressor-operator substituted with the analogue 7-deazaguanine.
Guanine residues in the lac operator were substituted with the isosteric analogue of 7-deazaguanine. The observed equilibrium dissociation constants for lac repressor binding to substituted operators were measured in 10 mM Tris, 150 mM KCl, 0.1 mM EDTA, and 0.1 mM DTE, pH 7.6, at 25 degrees C, using either a standard equilibrium assay or a competition assay. Of the seven individual sites tested, only three significantly altered the complex affinity. Two of these sites are symmetrically related and are positioned in the major grooves that are known to interact directly with repressor, and the third site is located in the central core region of the operator. The alkylation interference assay, which identifies essential phosphate sites, was used to define the extent of perturbation on the protein-DNA complex by the modified nucleotide. Chemical footprinting data for the singly substituted operator done at a single concentration of protein reflected the alignment of sterically incompatible groups or disruption in the local conformation, but did not appear to alter general phosphate backbone interactions. Similar experiments with a doubly substituted operator revealed that the free energy of binding was an additive combination of each of the individual sites, and the alkylation interference data were similar to the singly substituted and wild-type operators. A constitutive mutant which also had a higher binding constant had a similar alkylation interference pattern.
Zhang
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Department of Chemistry and Biochemistry, University of Delaware, Newark 19716.
Gottlieb
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PA
D000478:Alkylation; D001482:Base Composition; D001483:Base Sequence; D001667:Binding, Competitive; D004247:DNA / Q000378:metabolism*; D006147:Guanine / Q000031:analogs & derivatives*; D007700:Kinetics; D007763:Lac Operon / Q000187:drug effects; D008433:Mathematics; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides / Q000138:chemical synthesis / Q000378:metabolism* / Q000494:pharmacology; D012097:Repressor Proteins / Q000187:drug effects / Q000378:metabolism*; D013816:Thermodynamics
D009838:Oligodeoxyribonucleotides; D012097:Repressor Proteins; D006147:Guanine; D004247:DNA; C066856:7-deazaguanine