Quantitative Methods for Measuring DNA Flexibility In Vitro and In Vivo

The Escherichia coli lactose operon provides a paradigm for understanding gene control by DNA looping where the lac repressor (LacI) protein competes with RNA polymerase for DNA binding. Not all promoter loops involve direct competition between repressor and RNA polymerase. This raises the possibility that positioning a promoter within a tightly constrained DNA loop is repressive per se, an idea that has previously only been considered in vitro. Here, we engineer living E. coli bacteria to measure repression due to promoter positioning within such a tightly constrained DNA loop in the absence of protein–protein binding competition. We show that promoters held within such DNA loops are repressed ∼100-fold, with up to an additional ∼10-fold repression (∼1000-fold total) dependent on topological positioning of the promoter on the inner or outer face of the DNA loop. Chromatin immunoprecipitation data suggest that repression involves inhibition of both RNA polymerase initiation and elongation. These in vivo results show that gene repression can result from tightly looping promoter DNA even in the absence of direct competition between repressor and RNA polymerase binding.

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“…Expression constructs were confirmed by diagnostic polymerase chain reaction amplification following conjugation and selection (31). …”

Section: Methodsmentioning

confidence: 99%

Bacterial promoter repression by DNA looping without protein–protein binding competition

Becker

Greiner

Peters

et al. 2014

Nucleic Acids Research

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“…The repression ratio ( RR ) is given by the ratio of the raw induced luciferase activity divided by the repressed luciferase activity: The repression level, RL , is given by expression from the completely induced promoter in the absence of looping to the completely repressed promoter in the presence of looping While it is possible to extract fits to parameters related to DNA physical properties from repression data [11, 16, 18, 51–53], the present analysis of supercoiling effects on repression is presented qualitatively.…”

Section: Methodsmentioning

confidence: 99%

“…Evidence of looping facilitation by supercoiling has been reported in some in vitro experiments [28, 33]. On the other hand, the measured local stiffness of DNA in vitro [18, 34–37] limits the extent to which global supercoiling can increase local operator concentrations for short DNA loops that may be relevant to lac control in vivo. Here we devised an approach to explicitly test the hypothesis that negative DNA supercoiling facilitates the formation of small (6–8 DNA turns) repression loops in E .…”

Section: Introductionmentioning

confidence: 99%

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Supercoiling Effects on Short-Range DNA Looping in E. coli

2016

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DNA-protein loops can be essential for gene regulation. The Escherichia coli lactose (lac) operon is controlled by DNA-protein loops that have been studied for decades. Here we adapt this model to test the hypothesis that negative superhelical strain facilitates the formation of short-range (6–8 DNA turns) repression loops in E. coli. The natural negative superhelicity of E. coli DNA is regulated by the interplay of gyrase and topoisomerase enzymes, adding or removing negative supercoils, respectively. Here, we measured quantitatively DNA looping in three different E. coli strains characterized by different levels of global supercoiling: wild type, gyrase mutant (gyrB226), and topoisomerase mutant (ΔtopA10). DNA looping in each strain was measured by assaying repression of the endogenous lac operon, and repression of ten reporter constructs with DNA loop sizes between 70–85 base pairs. Our data are most simply interpreted as supporting the hypothesis that negative supercoiling facilitates gene repression by small DNA-protein loops in living bacteria.

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“…These experiments highlighted the modularity of the looping process in transcriptional regulation and launched this synthetic version of the lac operon as a platform from which to query the in vivo mechanical properties of DNA. [49, 50, 51]…”

Section: Bending the Lac Operon In Bacteriamentioning

confidence: 99%

Using synthetic biology to make cells tomorrow's test tubes

García

Brewster

Phillips

2016

Integrative Biology

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The main tenet of physical biology is that biological phenomena can be subject to the same quantitative and predictive understanding that physics has afforded in the context of inanimate matter. However, the inherent complexity of many of these biological processes often leads to the derivation of complex theoretical descriptions containing a plethora of unknown parameters. Such complex descriptions pose a conceptual challenge to the establishment of a solid basis for predictive biology. In this article, we present various exciting examples of how synthetic biology can be used to simplify biological systems and distill these phenomena down to their essential features as a means to enable their theoretical description. Here, synthetic biology goes beyond previous efforts to engineer nature and becomes a tool to bend nature to understand it. We discuss various recent and classic experiments featuring applications of this synthetic approach to the elucidation of problems ranging from bacteriophage infection, to transcriptional regulation in bacteria and in developing embryos, to evolution. In all of these examples, synthetic biology provides the opportunity to turn cells into the equivalent of a test tube, where biological phenomena can be reconstituted and our theoretical understanding put to test with the same ease that these same phenomena can be studied in the in vitro setting.

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Quantitative Methods for Measuring DNA Flexibility In Vitro and In Vivo

Cited by 28 publications

References 38 publications

Bacterial promoter repression by DNA looping without protein–protein binding competition

Bacterial promoter repression by DNA looping without protein–protein binding competition

Supercoiling Effects on Short-Range DNA Looping in E. coli

Using synthetic biology to make cells tomorrow's test tubes

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