Positive supercoiling favors transcription elongation through lac repressor-mediated DNA loops.

Some proteins, like the lac repressor (LacI), mediate long-range loops that alter DNA topology and create torsional barriers. During transcription, RNA polymerase generates supercoiling that may facilitate passage through such barriers. We monitored E. coli RNA polymerase progress along templates in conditions that prevented, or favored, 400 bp LacI-mediated DNA looping. Tethered particle motion measurements revealed that RNA polymerase paused longer at unlooped LacI obstacles or those barring entry to a loop than those barring exit from the loop. Enhanced dissociation of a LacI roadblock by the positive supercoiling generated ahead of a transcribing RNA polymerase within a torsion-constrained DNA loop may be responsible for this reduction in pause time. In support of this idea, RNA polymerase transcribed 6-fold more slowly through looped DNA and paused at LacI obstacles for 66% less time on positively supercoiled compared to relaxed templates, especially under increased tension (torque). Positive supercoiling propagating ahead of polymerase facilitated elongation along topologically complex, protein-coated templates.

Basic mechanisms and kinetics of pause-interspersed transcript elongation.

RNA polymerase pausing during elongation is an important mechanism in the regulation of gene expression. Pausing along DNA templates is thought to be induced by distinct signals encoded in the nucleic acid sequence and halt elongation complexes to allow time for necessary co-transcriptional events. Pausing signals have been classified as those producing short-lived elemental, long-lived backtracked, or hairpin-stabilized pauses. In recent years, structural microbiology and single-molecule studies have significantly advanced our understanding of the paused states, but the dynamics of these states are still uncertain, although several models have been proposed to explain the experimentally observed pausing behaviors. This review summarizes present knowledge about the paused states, discusses key discrepancies among the kinetic models and their basic assumptions, and highlights the importance and challenges in constructing theoretical models that may further our biochemical understanding of transcriptional pausing.

Nanomechanics of negatively supercoiled diaminopurine-substituted DNA.

Single molecule experiments have demonstrated a progressive transition from a B- to an L-form helix as DNA is gently stretched and progressively unwound. The particular sequence of a DNA segment defines both base stacking and hydrogen bonding that affect the partitioning and conformations of the two phases. Naturally or artificially modified bases alter H-bonds and base stacking and DNA with diaminopurine (DAP) replacing adenine was synthesized to produce linear fragments with triply hydrogen-bonded DAP:T base pairs. Both unmodified and DAP-substituted DNA transitioned from a B- to an L-helix under physiological conditions of mild tension and unwinding. This transition avoids writhing and the ease of this transition may prevent cumbersome topological rearrangements in genomic DNA that would require topoisomerase activity to resolve. L-DNA displayed about tenfold lower persistence length than B-DNA. However, left-handed DAP-substituted DNA was twice as stiff as unmodified L-DNA. Unmodified DNA and DAP-substituted DNA have very distinct mechanical characteristics at physiological levels of negative supercoiling and tension.

Negative DNA supercoiling makes protein-mediated looping deterministic and ergodic within the bacterial doubling time.

Protein-mediated DNA looping is fundamental to gene regulation and such loops occur stochastically in purified systems. Additional proteins increase the probability of looping, but these probabilities maintain a broad distribution. For example, the probability of lac repressor-mediated looping in individual molecules ranged 0-100%, and individual molecules exhibited representative behavior only in observations lasting an hour or more. Titrating with HU protein progressively compacted the DNA without narrowing the 0-100% distribution. Increased negative supercoiling produced an ensemble of molecules in which all individual molecules more closely resembled the average. Furthermore, in only 12 min of observation, well within the doubling time of the bacterium, most molecules exhibited the looping probability of the ensemble. DNA supercoiling, an inherent feature of all genomes, appears to impose time-constrained, emergent behavior on otherwise random molecular activity.

Macromolecular Crowding and DNA: Bridging the Gap between In Vitro and In Vivo.

The cellular environment is highly crowded, with up to 40% of the volume fraction of the cell occupied by various macromolecules. Most laboratory experiments take place in dilute buffer solutions; by adding various synthetic or organic macromolecules, researchers have begun to bridge the gap between in vitro and in vivo measurements. This is a review of the reported effects of macromolecular crowding on the compaction and extension of DNA, the effect of macromolecular crowding on DNA kinetics, and protein-DNA interactions. Theoretical models related to macromolecular crowding and DNA are briefly reviewed. Gaps in the literature, including the use of biologically relevant crowders, simultaneous use of multi-sized crowders, empirical connections between macromolecular crowding and liquid-liquid phase separation of nucleic materials are discussed.

Stability and change in autobiographical reasoning: A 4-year longitudinal study of narrative identity development.

OBJECTIVE: Research on personality development has traditionally focused on rank-order stability and mean-level change in the context of personality traits. The present study expands this approach to the examination of change and stability at another level of personality-narrative identity-by focusing on autobiographical reasoning. Drawing from theory in personality and developmental science, we examine stability and change in exploratory processing and positive and negative self-event connections. METHOD: We take advantage of a longitudinal study of emerging adult personality and identity development, which includes four waves of data across 4 years, examining reasoning in two domains of identity, academics, and romance (n = 1520 narratives; n = 176-638 participants, depending on the analysis). RESULTS: We found moderate rank-order stability in autobiographical reasoning, but more so for exploratory processing than self-event connections. We found mean-level increases for exploratory processing in the context of romance and stability in the context of academics. For self-event connections, we saw a decrease for positive connections, and for negative connections about romance, with stability for negative connections about academics. CONCLUSIONS: Implications include developmental differences in types of reasoning as well as the sensitivity of narrative identity to revealing the contextual nature of personality development.

Energetics of twisted DNA topologies.

Our goal is to review the main theoretical models used to calculate free energy changes associated with common, torsion-induced conformational changes in DNA and provide the resulting equations hoping to facilitate quantitative analysis of both in vitro and in vivo studies. This review begins with a summary of work regarding the energy change of the negative supercoiling-induced B- to L-DNA transition, followed by a discussion of the energetics associated with the transition to Z-form DNA. Finally, it describes the energy changes associated with the formation of DNA curls and plectonemes, which can regulate DNA-protein interactions and promote cross talk between distant DNA elements, respectively. The salient formulas and parameters for each scenario are summarized in table format to facilitate comparison and provide a concise, user-friendly resource.

Protein-mediated loops in supercoiled DNA create large topological domains.

Supercoiling can alter the form and base pairing of the double helix and directly impact protein binding. More indirectly, changes in protein binding and the stress of supercoiling also influence the thermodynamic stability of regulatory, protein-mediated loops and shift the equilibria of fundamental DNA/chromatin transactions. For example, supercoiling affects the hierarchical organization and function of chromatin in topologically associating domains (TADs) in both eukaryotes and bacteria. On the other hand, a protein-mediated loop in DNA can constrain supercoiling within a plectonemic structure. To characterize the extent of constrained supercoiling, 400 bp, lac repressor-secured loops were formed in extensively over- or under-wound DNA under gentle tension in a magnetic tweezer. The protein-mediated loops constrained variable amounts of supercoiling that often exceeded the maximum writhe expected for a 400 bp plectoneme. Loops with such high levels of supercoiling appear to be entangled with flanking domains. Thus, loop-mediating proteins operating on supercoiled substrates can establish topological domains that may coordinate gene regulation and other DNA transactions across spans in the genome that are larger than the separation between the binding sites.

Protein-mediated looping of DNA under tension requires supercoiling.

Protein-mediated DNA looping is ubiquitous in chromatin organization and gene regulation, but to what extent supercoiling or nucleoid associated proteins promote looping is poorly understood. Using the lac repressor (LacI), a paradigmatic loop-mediating protein, we measured LacI-induced looping as a function of either supercoiling or the concentration of the HU protein, an abundant nucleoid protein in Escherichia coli. Negative supercoiling to physiological levels with magnetic tweezers easily drove the looping probability from 0 to 100% in single DNA molecules under slight tension that likely exists in vivo. In contrast, even saturating (micromolar) concentrations of HU could not raise the looping probability above 30% in similarly stretched DNA or 80% in DNA without tension. Negative supercoiling is required to induce significant looping of DNA under any appreciable tension.

Nanomechanics of Diaminopurine-Substituted DNA.

2,6-diaminopurine (DAP) is a nucleobase analog of adenine. When incorporated into double-stranded DNA (dsDNA), it forms three hydrogen bonds with thymine. Rare in nature, DAP substitution alters the physical characteristics of a DNA molecule without sacrificing sequence specificity. Here, we show that in addition to stabilizing double-strand hybridization, DAP substitution also changes the mechanical and conformational properties of dsDNA. Thermal melting experiments reveal that DAP substitution raises melting temperatures without diminishing sequence-dependent effects. Using a combination of atomic force microscopy (AFM), magnetic tweezer (MT) nanomechanical assays, and circular dichroism spectroscopy, we demonstrate that DAP substitution increases the flexural rigidity of dsDNA yet also facilitates conformational shifts, which manifest as changes in molecule length. DAP substitution increases both the static and dynamic persistence length of DNA (measured by AFM and MT, respectively). In the static case (AFM), in which tension is not applied to the molecule, the contour length of DAP-DNA appears shorter than wild-type (WT)-DNA; under tension (MT), they have similar dynamic contour lengths. At tensions above 60 pN, WT-DNA undergoes characteristic overstretching because of strand separation (tension-induced melting) and spontaneous adoption of a conformation termed S-DNA. Cyclic overstretching and relaxation of WT-DNA at near-zero loading rates typically yields hysteresis, indicative of tension-induced melting; conversely, cyclic stretching of DAP-DNA showed little or no hysteresis, consistent with the adoption of the S-form, similar to what has been reported for GC-rich sequences. However, DAP-DNA overstretching is distinct from GC-rich overstretching in that it happens at a significantly lower tension. In physiological salt conditions, evenly mixed AT/GC DNA typically overstretches around 60 pN. GC-rich sequences overstretch at similar if not slightly higher tensions. Here, we show that DAP-DNA overstretches at 52 pN. In summary, DAP substitution decreases the overall stability of the B-form double helix, biasing toward non-B-form DNA helix conformations at zero tension and facilitating the B-to-S transition at high tension.

Model-free 3D localization with precision estimates for brightfield-imaged particles.

Volumetric imaging and 3D particle tracking are becoming increasingly common and have a variety of microscopy applications including in situ fluorescent imaging, in-vitro single-molecule characterization, and analysis of colloidal systems. While recent interest has generated discussion of optimal schemes for localizing diffraction-limited fluorescent puncta, there have been relatively few published routines for tracking particles imaged with bright-field illumination. To address this, we outline a simple, look-up-table based 3D tracking strategy, which can be adapted to most commercially available wide-field microscopes, and present two image processing algorithms that together yield high-precision localization and return estimates of statistical accuracy. Under bright-field illumination, a particle's depth can be determined based on the size and shape of its diffractive pattern due to Mie scattering. Contrary to typical "super-resolution" fluorescence tracking routines, which typically fit a diffraction-limited spot to a model point-spread-function, the lateral (XY) tracking routine relies on symmetry to locate a particle without prior knowledge of the form of the particle. At low noise levels (signal:noise > 1000), the symmetry routine estimates particle positions with accuracy better than 0.01 pixel. Depth localization is accomplished by matching images of particles to those in a pre-recorded look-up-table. The routine presented here optimally interpolates between LUT entries with better than 0.05 step accuracy. Both routines are tolerant of high levels of image noise, yielding sub-pixel/step accuracy with signal-to-noise ratios as small as 1, and, by design, return confidence intervals indicating the expected accuracy of each calculated position. The included implementations operate extremely quickly and are amenable to real-time analysis at frame rates exceeding several hundred frames per second.

DNA supercoiling: a regulatory signal for the λ repressor.

Topoisomerases, polymerases, and the chirality introduced by the binding of histones or nucleoid-associated proteins affect DNA supercoiling in vivo. However, supercoiling is not just a by-product of DNA metabolism. Supercoiling is an indicator of cell health, it modifies the accessibility of chromatin, and coordinates the transcription of genes. This suggests that regulatory, protein-mediated loops in DNA may sense supercoiling of the genome in which they are embedded. The λ repressor (CI) maintains the quiescent (lysogenic) transcriptome of bacteriophage λ in infected Escherichia coli. CI-mediated looping prevents overexpression of the repressor protein to preserve sensitivity to conditions that trigger virulence (lysis). Experiments were performed to assess how well the CI-mediated DNA loop traps superhelicity and determine whether supercoiling enhances CI-mediated DNA looping. CI oligomers partitioned plasmids into topological domains and prevented the passage of supercoiling between them. Furthermore, in single DNA molecules stretched and twisted with magnetic tweezers, levels of superhelical density confined in CI-mediated DNA loops ranged from -15% or +11%. Finally, in DNA under tensions that may occur in vivo, supercoiling lowered the free energy of loop formation and was essential for DNA looping. Supercoiling-enhanced looping can influence the maintenance of lysogeny in the λ repressor system; it can encode sensitivity to the energy level of the cell and creates independent topological domains of distinct superhelical density.

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

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 enhancer-promoter 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.

Quantitation of the DNA tethering effect in long-range DNA looping in vivo and in vitro using the Lac and λ repressors.

Efficient and specific interactions between proteins bound to the same DNA molecule can be dependent on the length of the DNA tether that connects them. Measurement of the strength of this DNA tethering effect has been largely confined to short separations between sites, and it is not clear how it contributes to long-range DNA looping interactions, such as occur over separations of tens to hundreds of kilobase pairs in vivo. Here, gene regulation experiments using the LacI and λ CI repressors, combined with mathematical modeling, were used to quantitate DNA tethering inside Escherichia coli cells over the 250- to 10,000-bp range. Although LacI and CI loop DNA in distinct ways, measurements of the tethering effect were very similar for both proteins. Tethering strength decreased with increasing separation, but even at 5- to 10-kb distances, was able to increase contact probability 10- to 20-fold and drive efficient looping. Tethering in vitro with the Lac repressor was measured for the same 600-to 3,200-bp DNAs using tethered particle motion, a single molecule technique, and was 5- to 45-fold weaker than in vivo over this range. Thus, the enhancement of looping seen previously in vivo at separations below 500 bp extends to large separations, underlining the need to understand how in vivo factors aid DNA looping. Our analysis also suggests how efficient and specific looping could be achieved over very long DNA separations, such as what occurs between enhancers and promoters in eukaryotic cells.

Influences of Exciton Diffusion and Exciton-Exciton Annihilation on Photon Emission Statistics of Carbon Nanotubes.

Pump-dependent photoluminescence imaging and second-order photon correlation studies have been performed on individual single-walled carbon nanotubes (SWCNTs) at room temperature. These studies enable the extraction of both the exciton diffusion constant and the Auger recombination coefficient. A linear correlation between these parameters is attributed to the effect of environmental disorder in setting the exciton mean free path and capture-limited Auger recombination at this length scale. A suppression of photon antibunching is attributed to the creation of multiple spatially nonoverlapping excitons in SWCNTs, whose diffusion length is shorter than the laser spot size. We conclude that complete antibunching at room temperature requires an enhancement of the exciton-exciton annihilation rate that may become realizable in SWCNTs allowing for strong exciton localization.

Single molecule analysis of DNA wrapping and looping by a circular 14mer wheel of the bacteriophage 186 CI repressor.

The lytic-lysogenic decision in bacteriophage 186 is governed by the 186 CI repressor protein in a unique way. The 186 CI is proposed to form a wheel-like oligomer that can mediate either wrapped or looped nucleoprotein complexes to provide the cooperative and competitive interactions needed for regulation. Although consistent with structural, biochemical and gene expression data, many aspects of this model are based on inference. Here, we use atomic force microscopy (AFM) to reveal the various predicted wrapped and looped species, and new ones, for CI regulation of lytic and lysogenic transcription. Automated AFM analysis showed CI particles of the predicted dimensions on the DNA, with CI multimerization favoured by DNA binding. Measurement of the length of the wrapped DNA segments indicated that CI may move on the DNA, wrapping or releasing DNA on either side of the wheel. Tethered particle motion experiments were consistent with wrapping and looping of DNA by CI in solution, where in contrast to λ repressor, the looped species were exceptionally stable. The CI regulatory system provides an intriguing comparison with that of nucleosomes, which share the ability to wrap and release similar sized segments of DNA.

Enhanced tethered-particle motion analysis reveals viscous effects.

Tethered-particle motion experiments do not require expensive or technically complex hardware, and increasing numbers of researchers are adopting this methodology to investigate the topological effects of agents that act on the tethering polymer or the characteristics of the polymer itself. These investigations depend on accurate measurement and interpretation of changes in the effective length of the tethering polymer (often DNA). However, the bead size, tether length, and buffer affect the confined diffusion of the bead in this experimental system. To evaluate the effects of these factors, improved measurements to calibrate the two-dimensional range of motion (excursion) versus DNA length were carried out. Microspheres of 160 or 240 nm in radius were tethered by DNA molecules ranging from 225 to 3477 basepairs in length in aqueous buffers containing 100 mM potassium glutamate and 8 mM MgCl2 or 10 mM Tris-HCl and 200 mM KCl, with or without 0.5% Tween added to the buffer, and the motion was recorded. Different buffers altered the excursion of beads on identical DNA tethers. Buffer with only 10 mM NaCl and >5 mM magnesium greatly reduced excursion. Glycerol added to increase viscosity slowed confined diffusion of the tethered beads but did not change excursion. The confined-diffusion coefficients for all tethered beads were smaller than those expected for freely diffusing beads and decreased for shorter tethers. Tethered-particle motion is a sensitive framework for diffusion experiments in which small beads on long leashes most closely resemble freely diffusing, untethered beads.

Distinct contributions of MSL complex subunits to the transcriptional enhancement responsible for dosage compensation in Drosophila.

The regulatory mechanism of dosage compensation is the paramount example of epigenetic regulation at the chromosomal level. In Drosophila, this mechanism, designed to compensate for the difference in the dosage of X-linked genes between the sexes, depends on the MSL complex that enhances the transcription of the single dose of these genes in males. We have investigated the function of various subunits of the complex in mediating dosage compensation. Our results confirm that the highly enriched specific acetylation of histone H4 at lysine 16 of compensated genes by the histone acetyl transferase subunit MOF induces a more disorganized state of their chromatin. We have determined that the association of the MSL complex reduces the level of negative supercoiling of the deoxyribonucleic acid of compensated genes, and we have defined the role that the other subunits of the complex play in this topological modification. Lastly, we have analyzed the potential contribution of ISWI-containing remodeling complexes to the architecture of compensated chromatin, and we suggest a role for this remodeling factor in dosage compensation.

Dividing a supercoiled DNA molecule into two independent topological domains.

Both prokaryotic and eukaryotic chromosomes are organized into many independent topological domains. These topological domains may be formed through constraining each DNA end from rotating by interacting with nuclear proteins; i.e., DNA-binding proteins. However, so far, evidence to support this hypothesis is still elusive. Here we developed two biochemical methods; i.e., DNA-nicking and DNA-gyrase methods to examine whether certain sequence-specific DNA-binding proteins are capable of separating a supercoiled DNA molecule into distinct topological domains. Our approach is based on the successful construction of a series of plasmid DNA templates that contain many tandem copies of one or two DNA-binding sites in two different locations. With these approaches and atomic force microscopy, we discovered that several sequence-specific DNA-binding proteins; i.e., lac repressor, gal repressor, and λ O protein, are able to divide a supercoiled DNA molecule into two independent topological domains. These topological domains are stable under our experimental conditions. Our results can be explained by a topological barrier model in which nucleoprotein complexes confine DNA supercoils to localized regions. We propose that DNA topological barriers are certain nucleoprotein complexes that contain stable toroidal supercoils assembled from DNA-looping or tightly wrapping DNA around DNA-binding proteins. The DNA topological barrier model may be a general mechanism for certain DNA-binding proteins, such as histone or histone-like proteins, to modulate topology of chromosome DNA in vivo.

Detecting DNA Loops Using Tethered Particle Motion.

The range of motion of a micron-sized bead tethered by a single polymer provides a dynamic readout of the effective length of the polymer. The excursions of the bead may reflect the intrinsic flexibility and/or topology of the polymer as well as changes due to the action activity of ligands that bind the polymer. This is a simple yet powerful experimental approach to investigate such interactions between DNA and proteins as demonstrated by experiments with the lac repressor. This protein forms a stable, tetrameric oligomer with two binding sites and can produce a loop of DNA between recognition sites separated along the length of a DNA molecule.