Designed hyperstable lac Repressor·DNA loop topologies suggest alternative loop geometries 1 1Edited by R. Ebright

Mehta, Ruchi A.; Kahn, Jason D.

doi:10.1006/jmbi.1999.3244

Cited by 57 publications

(128 citation statements)

References 35 publications

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“…Ruben and Roos (9) obtained electron microscopic images of freeze-etched samples of LacR that suggest an opening of the V in such a way that ␣, instead of being fixed at circa 34°, fluctuates about a value of 180°and the DNA binding sites lie at opposite ends of the complex. Measurements by Kahn and associates (10,11) of distances between fluorescent dyes attached to the ends of DNA constructs bound to LacR give strong evidence for the existence of these forms in solution. Atomic force microscopy images of 197-bp LacR-mediated loops recently observed by Virnik et al (12) on a 599-bp negatively supercoiled DNA minicircle are more appropriate, however, to state V as described above.…”

Section: Methodsmentioning

confidence: 98%

“…1). On the other hand, electron microscopy and solution studies (9)(10)(11)(12) indicate that the angle between the dimer subunits, i.e., the angle of aperture ␣, can vary. A change in ␣ affects the configuration of the DNA loop through its influence on the distance and orientation of the operators.…”

mentioning

confidence: 99%

“…Balaeff and coworkers (14) used their model to calculate the effect of linking number on loop energies and configurations. Kahn and coworkers (10,11) studied sequences other than the wild type and proposed models that account for the possibility of an extended LacR conformation with large angle of aperture. The models published to date do not account for the influence of DNA sequence and protein flexibility on the configuration of the O3-O1 loop as we do here.…”

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confidence: 99%

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Modeling the Lac repressor-operator assembly: The influence of DNA looping on Lac repressor conformation

Swigon

Coleman

Olson³

2006

Proc. Natl. Acad. Sci. U.S.A.

120

192

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Repression of transcription of the Escherichia coli Lac operon by the Lac repressor (LacR) is accompanied by the simultaneous binding of LacR to two operators and the formation of a DNA loop. A recently developed theory of sequence-dependent DNA elasticity enables one to relate the fine structure of the LacR-DNA complex to a wide range of heretofore-unconnected experimental observations. Here, that theory is used to calculate the configuration and free energy of the DNA loop as a function of its length and base-pair sequence, its linking number, and the end conditions imposed by the LacR tetramer. The tetramer can assume two types of conformations. Whereas a rigid V-shaped structure is observed in the crystal, EM images show extended forms in which two dimer subunits are flexibly joined. Upon comparing our computed loop configurations with published experimental observations of permanganate sensitivities, DNase I cutting patterns, and loop stabilities, we conclude that linear DNA segments of short-to-medium chain length (50 -180 bp) give rise to loops with the extended form of LacR and that loops formed within negatively supercoiled plasmids induce the V-shaped structure.lac operon ͉ sequence-dependent DNA elasticity ͉ DNase I footprinting M any genetic processes are controlled by proteins that bind at separate, often widely spaced, sites on DNA and hold the intervening double helix in a loop (1-3). The classical example is the lac operon of Escherichia coli (4). The Lac repressor (LacR) is a tetrameric protein assembly that represses the expression of the lac operon by simultaneously binding to two DNA sites, i.e., operators, in the vicinity of the nucleotides at which transcription starts. The structure and elastic properties of DNA determine which spacings of the operators are optimal for functionality. For the lac operon, a change in spacing by five to six nucleotides can induce a 50-fold alteration in the efficiency of repression (5, 6).Although there is a large amount of literature on genetic and biochemical aspects of expression in the lac system, less is known about the actual configuration of the LacR-DNA loop assembly. In the crystalline state the two dimer subunits of LacR are joined to form a V (7, 8), and contact with DNA is made at the tips of each arm of the V (Fig. 1). On the other hand, electron microscopy and solution studies (9-12) indicate that the angle between the dimer subunits, i.e., the angle of aperture ␣, can vary. A change in ␣ affects the configuration of the DNA loop through its influence on the distance and orientation of the operators. We are here concerned with loops formed between the primary operator site O1 and the weaker auxiliary site O3. As each operator binds to the protein in one of two possible orientations, there are four distinct loop types that are analogous to those considered by Geanacopoulos et al. (13) in their treatment of DNA loops in the E. coli gal operon. We write A1, A2, P1, and P2 for these loop types, where the A and P refer to antiparallel and parallel ...

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Section: Methodsmentioning

confidence: 98%

mentioning

confidence: 99%

mentioning

confidence: 99%

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Modeling the Lac repressor-operator assembly: The influence of DNA looping on Lac repressor conformation

Swigon

Coleman

Olson³

2006

Proc. Natl. Acad. Sci. U.S.A.

120

192

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“…Our focus has been to design hyperstable LacI-DNA loops with operator sequences bracketing sequence-directed bends, and then to use these molecules to probe loop structure and stability. [30][31][32] We made two constructs, denoted 9C14 and 11C12, that have different orientations of the lac operators with respect to sequence-directed curvature (Fig. 1).…”

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confidence: 99%

Flexibility and Control of Protein–dna Loops

Kahn

Cheong

Mehta

et al. 2006

Biophys. Rev. Lett.

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Protein-DNA loops are essential for efficient transcriptional repression and activation. The geometry and stability of the archetypal Lac repressor tetramer (LacI)-DNA loop were investigated using designed hyperstable loops containing lac operators bracketing a sequence-directed bend. Electrophoretic mobility shift assays, DNA cyclization, and bulk and single-molecule fluorescence resonance energy transfer (FRET) demonstrate that the DNA sequence controls whether the LacI-DNA loop forms a compact loop with positive writhe or an open loop with little writhe. Monte Carlo methods for simulation of DNA ring closure were extended to DNA loops, including treatment of variable protein hinge angles. The observed distribution of topoisomer products upon cyclization provides a strong constraint on possible models. The experiments and modeling imply that LacI-DNA can adopt a wide range of geometries but has a strong intrinsic preference for an open form. The flexibility of LacI helps explain in vivo observations that DNA looping is less sensitive to DNA length and shape than would be expected from the physical properties of DNA. While DNA cyclization suggests two pools of precursor loops for the 9C14 construct, single-molecule FRET demonstrates a single population. This discrepancy suggests that the LacI-DNA structure is strongly influenced by flanking DNA.

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“…The lac repressor can form loops of varied topology and stability as a result of the spacing of operators and the available conformations of the protein (Wong et al 2008) which may determine the disposition of other proteins that assemble on the loop (Mehta and Kahn 1999) and has been shown to regulate access by RNA polymerase (Becker et al 2014). In addition, accessory proteins that modify the intervening DNA by adding kinks, and/or altering flexibility, shift the looping equilibrium.…”

Section: Introductionmentioning

confidence: 99%

Supercoiling biases the formation of loops involved in gene regulation

Finzi

Dunlap

2016

Biophys Rev

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The function of DNA as a repository of genetic information is well-known. The post-genomic effort is to understand how this information-containing filament is chaperoned to manage its compaction and topological states. Indeed, the activities of enzymes that transcribe, replicate, or repair DNA are regulated to a large degree by access. Proteins that act at a distance along the filament by binding at one site and contacting another site, perhaps as part of a bigger complex, create loops that constitute topological domains and influence regulation. DNA loops and plectonemes are not necessarily spontaneous, especially large loops under tension for which high energy is required to bring their ends together, or small loops that require accessory proteins to facilitate DNA bending. However, the torsion in stiff filaments such as DNA dramatically modulates the topology, driving it from extended and genetically accessible to more looped and compact, genetically secured forms. Furthermore, there are accessory factors that bias the response of the DNA filament to supercoiling. For example, small molecules like polyamines, which neutralize the negative charge repulsions along the phosphate backbone, enhance flexibility and promote writhe over twist in response to torsion. Such increased flexibility likely pushes the topological equilibrium from twist toward writhe at tensions thought to exist in vivo. A predictable corollary is that stiffening DNA antagonizes looping and bending. Certain sequences are known to be more or less flexible or to exhibit curvature, and this may affect interactions with binding proteins. In vivo all of these factors operate simultaneously on DNA that is generally negatively supercoiled to some degree. Therefore, in order to better understand gene regulation that involves protein-mediated DNA loops, it is critical to understand the thermodynamics and kinetics of looping in DNA that is under tension, negatively supercoiled, and perhaps exposed to molecules that alter elasticity. Recent experiments quantitatively reveal how much negatively supercoiling DNA lowers the free energy of looping, possibly biasing the operation of genetic switches.

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Designed hyperstable lac Repressor·DNA loop topologies suggest alternative loop geometries 1 1Edited by R. Ebright

Cited by 57 publications

References 35 publications

Modeling the Lac repressor-operator assembly: The influence of DNA looping on Lac repressor conformation

Modeling the Lac repressor-operator assembly: The influence of DNA looping on Lac repressor conformation

Flexibility and Control of Protein–dna Loops

Supercoiling biases the formation of loops involved in gene regulation

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