lac repressor-operator interaction
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Lac repressor
Lactose permease
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Lac repressor
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Heterologous expression
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Lac repressor
Lactose permease
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Lac permease is a symport protein which responsible for the active accumulation of lactose in E. coli. The protein utilizes the energy from the downhill translocation of protons to drive uphill accumulation of lactose. This work reports the direct measurement by proton NMR of lactose binding to lac permease of E. coli membrane vesicles. The technique allows the determination of the K_d of lactose binding to lac permease binding sites. The results presented here show that the assay is specific for lac permease binding sites and that the assay can distinguish differences in binding affinity for different site specific mutants of lac permease. Determination of the Kd for lactose binding is important in determining which residues are important for lactose binding.
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The lac operon is a delicate inducible gene expression element in bacteria. To efficiently induce gene expression, a sufficient dosage of an inducer, usually that of 500–1000 µM isopropyl β‐D‐1‐thiogalactopyranoside (IPTG), is required to keep repressor LacI from its binding sites, which is a heavy cost burden in low‐value‐added products. So we propose a strategy to reduce the required dosage of IPTG by restricting LacI expression. To test this strategy, we employed a reconstructed IPTG inducible expression system based on lac operon, Promoter( lacO )‐target gene‐P tac L‐ lacI , where a modified promoter, P tac , with a random synthetic library (P tac L) to instead of P lacI to optimize LacI expression in Escherichia coli . Finally, the P tac L mutant, P tac L4, which could maintain the same repression effect as the original P lacI while reducing the required dosage of IPTG from 500 to 20 µM, was selected. This method is simple and efficient and can be of a good reference point for attempts to reduce inducer concentration in the IPTG or similar inducible expression systems.
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Lac repressor
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Bistability
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In vivo induction of the Escherichia coli lactose operon as a function of inducer concentration generates a sigmoidal curve, indicating a non-linear response. Suggested explanations for this dependence include a 2:1 inducer–repressor stoichiometry of induction, which is the currently accepted view. It is, however, known for decades that, in vitro , operator binding as a function of inducer concentration is not sigmoidal. This discrepancy between in vivo and in vitro data has so far not been resolved. We demonstrate that the in vivo non-linearity of induction is due to cooperative repression of the wild-type lac operon through DNA loop formation. In the absence of DNA loops, in vivo induction curves are hyperbolic. In the light of this result, we re-address the question of functional molecular inducer–repressor stoichiometry in induction of the lac operon.
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Lac repressor
gal operon
L-arabinose operon
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Abstract The role of the Escherichia coli lactose permease (LacY) in the homogeneous induction of the lactose‐inducible promoters P tac and P trc by the natural inducer lactose and the synthetic inducer isopropyl‐β‐ d ‐thiogalactopyranoside (IPTG) was investigated. Lactose requires active transport by LacY, whereas IPTG can freely penetrate the cell wall. In E. coli strains lacking a functional LacY, IPTG is required for induction of P tac and P trc . In E. coli strains carrying a functional LacY, induction of P trc and P tac with intermediate concentrations of lactose gave rise to two subpopulations, one fully induced and one uninduced, whereas a single, fully induced population resulted when high inducer concentrations were used. In contrast, induction with IPTG gave rise to a single population of cells at all inducer concentrations in both lacY and lacY + strains.
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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.
Lac repressor
Inducer
trp operon
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Lactose permease
Inducer
Strain (injury)
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