Induction of the lac promoter in the absence of DNA loops and the stoichiometry of induction

Oehler, Stefan; Alberti, Siegfried; Müller‐Hill, Benno

doi:10.1093/nar/gkj453

Cited by 44 publications

(62 citation statements)

References 42 publications

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“…3b is due to the enhanced dilution rate at saturating glucose concentrations. Reduction of the CRP-cAMP levels accounts for the remaining 2.5-fold decline, which is consistent with the data obtained by Kuhlman et al (Herzenberg, 1959) 0.007 mM for IPTG (Oehler et al, 2006) α 40 (Oehler et al, 1994) α 1200 (Oehler et al, 1994) α G 130 (Ozbudak et al, 2004) φ e /φ g See Eq. (25) φ s See Eq.…”

Section: The Effect Of Dilutionsupporting

confidence: 91%

The diffusive influx and carrier efflux have a strong effect on the bistability of the lac operon in Escherichia coli

Noel

Pilyugin

Narang

2009

Journal of Theoretical Biology

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In the presence of gratuitous inducers, the lac operon of Escherichia coli exhibits bistability. Most models in the literature assume that the inducer enters the cell via the carrier (permease), and exits by a diffusion-like process. The diffusive influx and carrier efflux are neglected. However, analysis of the data shows that in noninduced cells, the diffusive influx is comparable to the carrier influx, and in induced cells, the carrier efflux is 7 times the diffusive efflux. Since bistability entails the coexistence of steady states corresponding to both non-induced and induced cells, neither one of these fluxes can be ignored. Here, we formulate a model accounting for both fluxes. We show that: (a) The thresholds of bistability are profoundly affected by both fluxes. The diffusive influx reduces the on threshold by enhancing inducer accumulation in non-induced cells. The carrier efflux increases the off threshold by decreasing inducer accumulation in induced cells. (b) Simulations of the model with experimentally measured parameter values are in good agreement with the data for IPTG. However, there are discrepancies with respect to the data for TMG. They are most likely due to two features missing from the model, namely, the variation of the inducer exclusion effect and the specific growth rate with the lactose enzyme levels. (c) The steady states and thresholds obtained in the presence of both fluxes are well approximated by simple analytical expressions. These expressions provide a rigorous framework for the preliminary design of the lac genetic switch in synthetic biology.

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Section: The Effect Of Dilutionsupporting

confidence: 91%

The diffusive influx and carrier efflux have a strong effect on the bistability of the lac operon in Escherichia coli

Noel

Pilyugin

Narang

2009

Journal of Theoretical Biology

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“…The cooperativity produced by DNA looping generates a nonlinearity in the response of the system, so it can facilitate the appearance of bistability. Indeed, for the lac operon, the cooperativity introduced by the DNA loop (17,81) is responsible for the sigmoidicity of the induction curve of the response (82). Bistability was experimentally shown in early work on the lac operon (83) (but the term bistability was not used) and, more recently, by Ozbudak et al (82), looking at the expression of a lac-gfp reporter in individual cells.…”

Section: Attenuation Of Fluctuationsmentioning

confidence: 99%

DNA Looping in Prokaryotes: Experimental and Theoretical Approaches

Cournac

Plumbridge

2013

Journal of Bacteriology

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bTranscriptional regulation is at the heart of biological functions such as adaptation to a changing environment or to new carbon sources. One of the mechanisms which has been found to modulate transcription, either positively (activation) or negatively (repression), involves the formation of DNA loops. A DNA loop occurs when a protein or a complex of proteins simultaneously binds to two different sites on DNA with looping out of the intervening DNA. This simple mechanism is central to the regulation of several operons in the genome of the bacterium Escherichia coli, like the lac operon, one of the paradigms of genetic regulation. The aim of this review is to gather and discuss concepts and ideas from experimental biology and theoretical physics concerning DNA looping in genetic regulation. We first describe experimental techniques designed to show the formation of a DNA loop. We then present the benefits that can or could be derived from a mechanism involving DNA looping. Some of these are already experimentally proven, but others are theoretical predictions and merit experimental investigation. Then, we try to identify other genetic systems that could be regulated by a DNA looping mechanism in the genome of Escherichia coli. We found many operons that, according to our set of criteria, have a good chance to be regulated with a DNA loop. Finally, we discuss the proposition recently made by both biologists and physicists that this mechanism could also act at the genomic scale and play a crucial role in the spatial organization of genomes. Different levels of DNA organization exist within bacterial chromosomes. In the case of Escherichia coli, the genome has been shown to be organized, on the largest scale, in four individual macrodomains (Ter, Ori, Right, and Left) and two less-structured regions (1) that have a precise localization within the cell throughout the cell cycle and are associated with specific binding proteins (2). Large-scale DNA loops have been visualized by nucleoidspreading techniques and are thought to be stabilized by membrane and/or RNA components (3, 4). Then, at the scale of 10 kb, there are topological domains formed by supercoiled structures (5, 6) whose barriers are not placed stably at fixed sites but instead are randomly distributed (7). These intermediate loops can be stabilized with nucleoid-associated proteins like H-NS (8). Finally, there are smaller loops of a few hundred base pairs made by specific transcription factors that have a direct impact on transcription. Although loops of different sizes can have functional consequences for genomic organization and genetic regulation, it is the last category that we focus on in this review.A first hint that a transcription factor can bind simultaneously to two sites derived from the work of Kania and Müller-Hill in 1977 (9). However, the first experimental demonstration and clear proposal for the existence of a DNA loop affecting gene regulation was in 1984, by the team of Robert Schleif (10), working on the regulatory region of the ara...

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“…Because inducer binding is relatively weak-the equilibrium dissociation constant (K d ) is ϳ7 M for isopropyl-␤-D-thiogalactopyranoside (IPTG) (13)-and repressor concentration in wt cells is low (ϳ10 nM), it can only be used for strains overexpressing LacR or for preparations enriched in repressor (4,10). On the other hand, the affinity of repressor to operator is high (K d of ϳ10 pM in vitro for symmetric "ideal" lac operator) (13,18,20), which suggests stoichiometric titration of lac operator as an alternative approach.…”

mentioning

confidence: 99%

“…All binding reactions were done on ice for 15 min in volumes of 10 or 20 l BBϩ with 1 nM of the DNA fragment. The products of the binding reaction were assayed with band shift assays as described previously (13). Figure 2A shows the quantitative evaluation.…”

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confidence: 99%

Feedback Regulation of Lac Repressor Expression inEscherichia coli

Oehler

2009

J Bacteriol

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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.The lac operon is one of the classical model systems for transcriptional regulation (9). Expression of the tricistronic lac mRNA is negatively controlled (repressed) by the operon-specific Lac repressor (LacR) and positively controlled (activated) by the global regulator CAP (17). In recent years, the lac operon has become a focus and testing ground for systems biology analyses (19). Such modeling can only deliver meaningful results when it incorporates all relevant features of the system. The textbook picture of the lac operon has sustained substantial change over the years (9), and despite the wealth of information, many questions are still unanswered.

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Induction of the lac promoter in the absence of DNA loops and the stoichiometry of induction

Cited by 44 publications

References 42 publications

The diffusive influx and carrier efflux have a strong effect on the bistability of the lac operon in Escherichia coli

The diffusive influx and carrier efflux have a strong effect on the bistability of the lac operon in Escherichia coli

DNA Looping in Prokaryotes: Experimental and Theoretical Approaches

Feedback Regulation of Lac Repressor Expression inEscherichia coli

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