Structural dynamics of the lac repressor–DNA complex revealed by a multiscale simulation

The free energy of looping DNA by proteins and protein complexes determines to what extent distal DNA sites can affect each other. We inferred its in vivo value through a combined computationalexperimental approach for different lengths of the loop and found that, in addition to the intrinsic periodicity of the DNA double helix, the free energy has an oscillatory component of about half the helical period. Moreover, the oscillations have such an amplitude that the effects of regulatory molecules become strongly dependent on their precise DNA positioning and yet easily tunable by their cooperative interactions. These unexpected results can confer to the physical properties of DNA a more prominent role at shaping the properties of gene regulation than previously thought.computational modeling ͉ DNA looping ͉ gene expression ͉ lac operon ͉ regulation T he cell is a densely packed dynamic structure made of thousands of different molecular species that orchestrate their interactions to form a functional unit. Such complexity poses a strong barrier for experimentally characterizing the cellular components: not only the properties of the components can change when studied in vitro outside the cell, but also the in vivo probing of the cell can perturb the process under study (1). Here we use computational modeling to obtain the properties of the in vivo unperturbed components at the molecular level from physiological measurements at the cellular level. Explicitly, we infer the in vivo free energies of DNA looping from enzyme production in the lac operon (2).The formation of DNA loops by the binding of proteins at distal DNA sites plays a fundamental role in many cellular processes, such as transcription, recombination, and replication (3-5). In gene regulation, proteins bound far away from the genes they regulate can be brought to the initiation of transcription region by looping the intervening DNA. The free energy cost of this process determines how easily DNA can loop and therefore the extent to which distal DNA sites can affect each other (5).In the lac operon, there is a repressor molecule that regulates transcription by binding specifically to DNA sites known as operators and preventing the RNA polymerase from transcribing the genes. DNA looping allows the repressor to bind to two operators simultaneously, leading to an increase in repression of transcription. This increase, characterized by the repression level, can be connected to the free energy of looping DNA by a recent model for transcription regulation by the lac repressor (6). The distance between operators determines the length of the DNA loop and affects the repression level through the changes in the free energy of looping. For interoperator distances from 57.5 to 98.5 bp, Muller et al. (7) systematically varied the distance between two operators in increments of 1 bp and measured the in vivo repression levels under conditions similar to wild type. These physiological measurements of enzyme production in Escherichia coli cell populations allowed us to ...

Section: Appendix Ii: Corroboration Of the Main Results)mentioning

confidence: 99%

Inferring the in vivo looping properties of DNA

Saiz

Rubı́

Vilar

2005

“…Because the internal loop brings the CI sites 3.3-fold closer together (2,000/600 bp), we expected that the distance-shortening effect alone would give α = 3.3 1.5 = 6. The higher assistance seen may be due to specific angles imposed on the two 300-bp DNA "arms" as they exit the internal LacI tetramer (65), which, combined with the relative stiffness of DNA in vitro (persistence length ∼150 bp) (22), may tend to juxtapose the CI sites.…”

Section: Discussionmentioning

confidence: 99%

Quantitation of interactions between two DNA loops demonstrates loop domain insulation in E. coli cells

Priest

Kumar²,

Yan³

et al. 2014

Eukaryotic gene regulation involves complex patterns of long-range DNA-looping interactions between enhancers and promoters, but how these specific interactions are achieved is poorly understood. Models that posit other DNA loops-that aid or inhibit enhancerpromoter contact-are difficult to test or quantitate rigorously in eukaryotic cells. Here, we use the well-characterized DNA-looping proteins Lac repressor and phage λ CI to measure interactions between pairs of long DNA loops in E. coli cells in the three possible topological arrangements. We find that side-by-side loops do not affect each other. Nested loops assist each other's formation consistent with their distance-shortening effect. In contrast, alternating loops, where one looping element is placed within the other DNA loop, inhibit each other's formation, thus providing clear support for the loop domain model for insulation. Modeling shows that combining loop assistance and loop interference can provide strong specificity in long-range interactions.Lac repressor | lambda CI | tethered particle motion | statistical mechanical modeling T ranscription of genes is regulated by promoter-proximal DNA elements and distal DNA elements that together determine condition-dependent gene expression. In eukaryotic genomes, enhancers can be many hundreds of kilobases away from the promoter they regulate (1-3), and the intervening DNA can contain other promoters and other enhancers (4-7). How the regulatory influence of distal elements is exerted efficiently and specifically at the correct promoters is poorly understood.Enhancers are clusters of binding sites for transcription factors and chromatin-modifying enzymes, and activate promoters by directly contacting them via DNA looping (8-12). Enhancer trap approaches and mapping of transcription factor binding and chromatin modifications have identified tens of thousands of enhancer elements in metazoan genomes (7,(13)(14)(15)(16). Chromatin capture studies show that enhancers and promoters are connected in highly complex condition-dependent patterns (6,15,17). Although core enhancer and promoter elements can provide some specificity (18), enhancers are often able to activate heterologous promoters if they are placed near to each other. Indeed, this lack of specificity is the basis for standard enhancer assays and screens (7,14,19). Thus, additional mechanisms are clearly needed to target enhancers to the correct promoters over long distances and to prevent their interaction with the wrong promoters. Dedicated DNA-looping elements that can either assist or interfere with enhancer-promoter looping are thought to play a major role.In theory, any DNA loop that brings the enhancer and promoter closer together should assist their interaction (Fig. 1A), because the efficiency of contact increases as the length of the DNA tether between the sites shortens (20-24). Promoter-tethering elements in Drosophila that allow activation by specific enhancers over long distances are proposed to form DNA loops between sequences near the ...

“…The ability to calculate the loop free energy for any DNA sequence makes it possible to address the role of motions of DNA headpieces in looping, as suggested by recent molecular dynamics simulations (33), and the issue of the experimentally observed dependence of repression efficiency on the number, quality, and distance of operators (e.g., refs. 5, 6, 32, and 34).…”

Section: Discussionmentioning

confidence: 99%

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

Swigon

Coleman

Olson³

2006

119

192

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 ...