Lactose repressor hinge domain independently binds DNA
Joseph S. XuMadeleine N. HewittJaskeerat S. GulatiMatthew A. CruzHongli ZhanShirley LiuKathleen S. Matthews
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Abstract:
The short 8-10 amino acid "hinge" sequence in lactose repressor (LacI), present in other LacI/GalR family members, links DNA and inducer-binding domains. Structural studies of full-length or truncated LacI-operator DNA complexes demonstrate insertion of the dimeric helical "hinge" structure at the center of the operator sequence. This association bends the DNA ∼40° and aligns flanking semi-symmetric DNA sites for optimal contact by the N-terminal helix-turn-helix (HtH) sequences within each dimer. In contrast, the hinge region remains unfolded when bound to nonspecific DNA sequences. To determine ability of the hinge helix alone to mediate DNA binding, we examined (i) binding of LacI variants with deletion of residues 1-50 to remove the HtH DNA binding domain or residues 1-58 to remove both HtH and hinge domains and (ii) binding of a synthetic peptide corresponding to the hinge sequence with a Val52Cys substitution that allows reversible dimer formation via a disulfide linkage. Binding affinity for DNA is orders of magnitude lower in the absence of the helix-turn-helix domain with its highly positive charge. LacI missing residues 1-50 binds to DNA with ∼4-fold greater affinity for operator than for nonspecific sequences with minimal impact of inducer presence; in contrast, LacI missing residues 1-58 exhibits no detectable affinity for DNA. In oxidized form, the dimeric hinge peptide alone binds to O1 and nonspecific DNA with similarly small difference in affinity; reduction to monomer diminished binding to both O1 and nonspecific targets. These results comport with recent reports regarding LacI hinge interaction with DNA sequences.Keywords:
Lac repressor
Helix (gastropod)
Negative feedback regulation, mediated through repressor binding site O3, which overlaps the lacI gene, could explain the robustness of the weak expression of Lac repressor. Significant autorepression of Lac repressor has never been ruled out. In the work presented here, the degree of autoregulation of Lac repressor was determined. It is negligible.
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Recognition of the lac operator by the lac repressor involves specific interactions between residues in the repressor's recognition helix and bases in the DNA major groove. Tyr17 and Gln18, at positions 1 and 2 in the lac repressor recognition helix, can be exchanged for other amino acids to generate mutant repressors that display altered specificity. We have solved the solution structure of a protein-DNA complex of an altered-specificity mutant lac headpiece in which Tyr17 and Gln18 were exchanged for valine and alanine, respectively, as found in the recognition helix of the gal repressor. As previously described by Lehming et al. (EMBO J. 1987, 6, 3145-3153), this altered-specificity mutant of the lac repressor recognizes a variant lac operator that is similar to the gal operator Oe. The mutant lac headpiece showed the predicted specificity and is also able to mimic the gal repressor by recognizing and bending the natural gal operator Oe. These structural data show that, while most of the anchoring points that help the lac headpiece to assemble on the lac operator were preserved, a different network of protein-DNA interactions connecting Ala17 and Val18 to bases in the DNA major groove drives the specificity towards the altered operator.
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The lac operon of Escherichia coli is the paradigm for gene regulation. Its key component is the lac repressor, a product of the lacI gene. The three-dimensional structures of the intact lac repressor, the lac repressor bound to the gratuitous inducer isopropyl-β-D-1-thiogalactoside (IPTG) and the lac repressor complexed with a 21-base pair symmetric operator DNA have been determined. These three structures show the conformation of the molecule in both the induced and repressed states and provide a framework for understanding a wealth of biochemical and genetic information. The DNA sequence of the lac operon has three lac repressor recognition sites in a stretch of 500 base pairs. The crystallographic structure of the complex with DNA suggests that the tetrameric repressor functions synergistically with catabolite gene activator protein (CAP) and participates in the quaternary formation of repression loops in which one tetrameric repressor interacts simultaneously with two sites on the genomic DNA.
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Lac repressor
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Lac repressor
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trp operon
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Lac repressor
Enzyme Repression
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gal operon
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For many years the lac operon of Escherichia coli has been the paradigm for gene regulation. Recently, the structures of the lac repressor core bound to isopropyl-β-D-1-thiogalactoside (IPTG), the intact apo lac repressor, the intact lac repressor complexes with IPTG and a 21-base-pair symmetric operator, and the refined headpiece of the repressor have been determined. These structures have provided a framework for understanding a wealth of biochemical and genetic information. An analysis of these structures, as well as a description of their function and a comparison to homologous proteins, is now possible.
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Lac repressor
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Gillard, N., Spotheim-Maurizot, M. and Charlier, M. Radiation Abolishes Inducer Binding to Lactose Repressor. Radiat. Res. 163, 433–446 (2005).The lactose operon functions under the control of the repressor-operator system. Binding of the repressor to the operator prevents the expression of the structural genes. This interaction can be destroyed by the binding of an inducer to the repressor. If ionizing radiations damage the partners, a dramatic dysfunction of the regulation system may be expected. We showed previously that γ irradiation hinders repressor-operator binding through protein damage. Here we show that irradiation of the repressor abolishes the binding of the gratuitous inducer isopropyl-1-β-d-thiogalactoside (IPTG) to the repressor. The observed lack of release of the repressor from the complex results from the loss of the ability of the inducer to bind to the repressor due to the destruction of the IPTG binding site. Fluorescence measurements show that both tryptophan residues located in or near the IPTG binding site are damaged. Since tryptophan damage is strongly correlated with the loss of IPTG binding ability, we conclude that it plays a critical role in the effect. A model was built that takes into account the kinetic analysis of damage production and the observed protection of its binding site by IPTG. This model satisfactorily accounts for the experimental results and allows us to understand the radiation-induced effects.
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trp operon
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