In vivo interaction of Escherichia coli lac repressor N-terminal fragments with the lac operator

Khoury, Anastasia M.; Nick, Harry S.; Lu, Ponzy

doi:10.1016/0022-2836(91)90659-t

Cited by 33 publications

(21 citation statements)

References 39 publications

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“…2). The effects observed for 2AP or P nucleotides base paired with uracil at these positions are entirely consistent with this interpretation ( (42,43). In general, the wild-type interaction has demonstrated asymmetry mutations and substitutions in the left half of the operator always result in greater changes in affinity than those in the right half (9,14) and crosslinking experiments underscore the differences between the two halves (44).…”

Section: Direct Interaction Of Major Groove Sites In the Operatormentioning

confidence: 54%

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

Zhang

Gottlieb

1995

Nucl Acids Res

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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.

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Section: Direct Interaction Of Major Groove Sites In the Operatormentioning

confidence: 54%

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

Zhang

Gottlieb

1995

Nucl Acids Res

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“…The DNA binding domain of the lac repressor monomers is located at the N-terminal part (the ''headpiece'') and comprises approximately the first 60 amino acids (Markiewicz et al, 1994). From structural and biochemical studies performed on this DNA binding domain it has been demonstrated that this isolated headpiece retains its three dimensional structure and its specific DNA binding properties (Geisler & Weber, 1977;Ogata & Gilbert, 1979;Khoury et al, 1991). Previous studies on the structure of lac headpiece (Zuiderweg et al, 1983;Kaptein et al, 1985;Boelens et al, 1987;de Vlieg et al, 1988) have revealed that the protein contains three a-helices both in the free and DNA-bound state.…”

Section: Introductionmentioning

confidence: 99%

Refined Structure of Repressor Headpiece (1-56) Determined by Relaxation Matrix Calculations from 2D and 3D NOE Data: Change of Tertiary Structure upon Binding to the Operator

Slijper

Bonvin

Boelens

et al. 1996

Journal of Molecular Biology

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“…1) (7)(8)(9)(10). The N-terminal 60 amino acids in each monomer form a helix-turn-helix motif that directly contacts the operator DNA (7,(11)(12)(13)(14)(15)(16)(17). When isolated, this region exhibits site-specific DNA binding, albeit with significantly lower affinity than the intact protein (11,18,19).…”

mentioning

confidence: 99%

Glycine Insertion in the Hinge Region of Lactose Repressor Protein Alters DNA Binding

Falcon

Matthews

1999

Journal of Biological Chemistry

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Amino acid alterations were designed at the C terminus of the hinge segment (amino acids ϳ51-59 The lac repressor (LacI) negatively regulates lac mRNA synthesis by binding with high affinity to its target operator site and thereby precluding transcription by RNA polymerase (1-6). In response to the presence of inducer sugars, LacI undergoes a conformational change that results in diminished affinity for operator sequences without effect on nonspecific DNA binding (1, 2, 7). LacI is a protein of 150,000 Da composed of four identical subunits assembled as a dimer of dimers (see Fig.

show abstract

In vivo interaction of Escherichia coli lac repressor N-terminal fragments with the lac operator

Cited by 33 publications

References 39 publications

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

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

Refined Structure of Repressor Headpiece (1-56) Determined by Relaxation Matrix Calculations from 2D and 3D NOE Data: Change of Tertiary Structure upon Binding to the Operator

Glycine Insertion in the Hinge Region of Lactose Repressor Protein Alters DNA Binding

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