Modified nucleotides reveal the indirect role of the central base pairs in stabilizing thelacrepressor-operator complex

Abstract

Guanine residues in the lac operator were replaced by 2-aminopurine or purine analogues, pairing the modified nucleotides with C. The observed equilibrium dissociation constants for lac repressor binding to substituted operators were measured in 10 mM Tris, 150 mM KCI, 0.1 mM EDTA, 0.1 mM DTE, pH 7.6 at 250C. 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-aminopurine 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 Iobs of the complex was then measured when 2-aminopurine or purine analogues were paired with uracil 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 op 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 DNA 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.

RNA Structure Inhibits the TRAP ( rp RNA-binding AttenuationProtein)-RNA Interaction

“…Second, there is no correlation between the position of C7 G in the repeat and its effect, because enhanced affinity for TRAP is observed with ((G/ U)AGCC) 11 RNAs that contain C7 G but not with (GAGUU) 11 RNA. This effect of C7 G on the affinity of the TRAP-RNA complex, by perturbing the structure of the nucleic acid, is similar to observations with EcoRI (31) and the lac repressor (32). Incorporation of modified nucleotides in the target DNAs of these proteins also enhances their enzymatic and/or binding activities by affecting the structures of these DNAs.…”

Strong DNA binding by covalently linked dimeric Lac headpiece: Evidence for the crucial role of the hinge helices

“…Therefore, the instability of the hinge helices in the right half-site originates only in the local contact of the protein with the minor groove and is most probably related to the absence of hydrogen bonding between the backbone of Asn-50 and DNA. Our results confirm previous biochemical data indicating that the central base pairs may modulate the binding of the repressoroperator complex by altering the structure and flexibility of DNA (27). Therefore, the DNA phosphate conformation in the minor groove appears to be optimal for hydrogen bonding to Asn-50 only in the left site region.…”

Determinants of Bacteriophage 933W Repressor DNA Binding Specificity

“…The affinities of these sites for 933W repressor were measured by competition with the consensus binding site (Methods and Materials, [14], [21]) in an electrophoresis mobility shift assay. In a direct binding EMSA experiment, the mixture of protein and DNA is not at chemical equilibrium during the electrophoresis.…”

“…These experiments were performed as essentially described [14]. Briefly, following isolation from a polyacrylamide gel, the oligonucleotide encoding the 933W consensus binding site sequence was radioactively labeled at its 5′ ends as described above.…”