Nucleic Acids Research, 1995, Vol. 23, No. 9 1502-1511
© 1995
MOLECULAR BIOLOGY |
Modified nucleotides reveal the indirect role of the central base pairs in stabilizing the lac repressor-operator complex
Department of Chemistry and Biochemistry, University of Delaware Newark, DE 19716, USA
*To whom correspondence should be addressed
Received February 3, 1995. Revised March 13, 1995. Accepted March 13, 1995.
Guanine residues in the lac operator were replaced by 2-aminopurlne or purlne analogues, pairing the modified nucleotides with C. The observed equilibrium dissociation constants for lac repressor binding to substituted operators were measured In 10 mM Trls, 150 mM KCI, 0.1 mM EDTA, 0.1 mM DTE, pH 7.6 at 25°C. These measurements revealed five positions that destabilized the complex when substituted with either analogue. Two positions, which are related by a 2-fold symmetry, are in the major groove of the operator thought to directly interact with the protein. Three sites were In the central region of the operator. A purine analogue at a sixth site perturbed the local DNA structure and destabilized the complex. Alkylation interference experiments of the 2-amlnopurine substituted operators demonstrated that, of the five affected, two substitutions displayed altered phosphate interference patterns at the phosphate adjacent to the substituted base. For these operators, complex formation was measured in different concentrations of KCI to assess the contribution of counterion release to the bimolecular process. The results Indicated that both complexes were similar to wild-type, although minor changes were observed. The Kobs of the complex was then measured when 2-aminopurlne or purine analogues were paired with uracll nucleotide, a base pair that serves to stabilize the DNA. The introduction of the new base pairs revealed two effects on the bimolecular interaction. For those operator sites that are thought to perturb the interaction directly, the affinity of the complex was weakened to levels observed for the singly-substituted operators. In contrast, the nucleotides of 2-aminopurine paired with uracil positioned In the central region of the operator served to enhance the stability of the complex. The purine-uracil base pair substitution on the other hand had a significant destabilizing effect on the interaction. We propose that the central base pairs modulate binding of the complex by altering the intrinsic properties of the DNA. Two specific attributes are required to achieve the lowest free energy of interaction. The D[NA must have two Interstrand hydrogen bonds to stabilize the duplex and it must have properties associated with directional bending or unwinding. This analysis does not rule out contributions by direct interactions between the protein and the central region of the operator but underscores how Indirect effects play a major role In complex formation in this system.
+Present address: Nucleic Acid Facility, University of Pennsylvania Cancer Center, Philadelphia, PA 19104, USA
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