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DNA can act as a scaffold for the cooperative binding of protein oligomers. For example, the phage 186 CI repressor forms a wheel of seven dimers wrapped in DNA with specific binding sites, while phage λ CI repressor dimers bind to two well-separated sets of operators, forming a DNA loop. Atomic force microscopy was used to measure transcription elongation by Escherichia coli RNA polymerase (RNAP) through these protein complexes. 186 CI, or λ CI, bound along unlooped DNA negligibly interfered with transcription by RNAP. Wrapped and looped topologies induced by these scaffolded, cooperatively bound repressor oligomers did not form significantly better roadblocks to transcription. Thus, despite binding with high affinity, these repressors are not effective roadblocks to transcription.

During transcription, RNA polymerases (RNAPs) must transit through roadblocks of various strengths to produce biologically meaningful transcripts. The roadblocking strength of DNA binding proteins varies significantly according to their affinity for DNA. Here we demonstrate that Escherichia coli RNAP can adopt a hybrid mechanism consisting of a passive and an active pathway to effectively transit through two roadblocks of different strengths, the lac repressor protein and EcoRI Q111. The passive pathway, where RNAP waits for the dissociation of the roadblock, is efficient against relatively weak roadblocks. The active mechanism, where RNAP dislodges the roadblock by repetitive cycles of backtracking and recovery, is essential for transit through strong roadblocks. Force direction and transcription factor GreA can bias the choice between the two pathways and modulate the rate of passage through roadblocks. These results further our understanding of how RNAPs transit through roadblocks.

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.

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.

Transcriptional pausing is highly regulated by the template DNA and nascent transcript sequences. Here, we propose a thermodynamic model of transcriptional pausing, based on the thermal energy of transcription bubbles and nascent RNA structures, to describe the kinetics of the reaction pathways between active translocation, elemental, backtracked, and hairpin-stabilized pauses. The model readily predicts experimentally detected pauses in high-resolution optical-tweezer measurements of transcription. Unlike other models, it also predicts the effect of tension and the GreA transcription factor on pausing.

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.

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.

During transcription, RNA polymerase (RNAP) translocates along the helical template DNA while maintaining high transcriptional fidelity. However, all genomes are dynamically twisted, writhed, and decorated by bound proteins and motor enzymes. In prokaryotes, proteins bound to DNA, specifically or not, frequently compact DNA into conformations that may silence genes by obstructing RNAP. Collision of RNAPs with these architectural proteins, may result in RNAP stalling and/or displacement of the protein roadblock. It is important to understand how rapidly transcribing RNAPs operate under different levels of supercoiling or in the presence of roadblocks. Given the broad range of asynchronous dynamics exhibited by transcriptional complexes, single-molecule assays, such as atomic force microscopy, fluorescence detection, optical and magnetic tweezers, etc. are well suited for detecting and quantifying activity with adequate spatial and temporal resolution. Here, we summarize current understanding of the effects of torsion and roadblocks on prokaryotic transcription, with a focus on single-molecule assays that provide real-time detection and readout.

DNA can act as a scaffold for the cooperative binding of protein oligomers. For example, the phage 186 CI repressor forms a wheel of seven dimers wrapped in DNA carrying specific binding sites, while the phage λ CI repressor binds in units of dimers to two well-separated sets of operators, forming a DNA loop. Atomic force microscopy was used to measure transcription elongation by E. coli RNA polymerase through these protein complexes. 186 CI or λ CI bound along unlooped DNA negligibly interfered with transcription by RNAP. More complex topologies induced by scaffolded, cooperatively bound repressor oligomers did not form significantly better roadblocks to transcription. Thus, despite binding with rather high affinity, these repressors are not effective roadblocks to transcription.

The histone-like protein from E. coli strain U93 (HU) is an abundant nucleoid-associated protein that contributes to the compaction of the bacterial genome as well as to the regulation of many of its transactions. Despite many years of investigations, the way and extent to which HU binding alters the DNA double helix and/or generates hierarchical structures using DNA as a scaffold is not completely understood. Here we combined single-molecule magnetic measurements with circular dichroism studies to monitor structural changes in the DNA-HU fiber as HU concentration was increased from 0 to 1000 nM under low and physiological monovalent salt conditions. We confirmed that DNA compaction correlated with HU concentration in a biphasic manner but DNA unwinding varied monotonically with HU concentration in 100 mM KCl. Instead, in more physiological 200 mM salt conditions, DNA compaction was monotonic while HU-induced DNA unwinding was negligible. Differential compaction and unwinding of DNA may be part of the response of bacteria to large variations in salt concentrations.

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 twelve minutes 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.

Single molecule experiments have demonstrated a progressive transition from a B- to an L-form helix as DNA is gently stretched and progressively unwound. Since the particular sequence of a DNA segment influences both base stacking and hydrogen bonding, the conformational dynamics of B-to-L transitions should be tunable. To test this idea, DNA with diaminopurine replacing adenine was synthesized to produce linear fragments with triply hydrogen-bonded A:T base pairs. Triple hydrogen bonding stiffened the DNA by 30% flexurally. In addition, DAP-substituted DNA formed plectonemes with larger gyres for both B- and L-form helices. 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 by DNA stretched and unwound by enzymatic activity. The intramolecular nature and ease of this transition likely prevent cumbersome topological rearrangements in genomic DNA that would require topoisomerase activity to resolve. L-DNA displayed about tenfold lower persistence length indicating it is much more contractile and prone to sharp bends and kinks. However, left-handed DAP DNA was twice as stiff as unmodified L-DNA. Thus, significantly doubly and triply hydrogen bonded segments have very distinct mechanical dynamics at physiological levels of negative supercoiling and tension.

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.

Transcriptional pausing is highly regulated by the template DNA and nascent transcript sequences. Here, we propose a thermodynamic model of transcriptional pausing, based on the thermal energy of transcription bubbles and nascent RNA structures, to describe the kinetics of the reaction pathways between active translocation, intermediate, backtracked, and hairpin-stabilized pauses. The model readily predicts experimentally detected pauses in high-resolution optical tweezers measurements of transcription. Unlike other models, it also predicts the effect of tension and the GreA transcription factor on pausing.

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 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.

Force spectroscopy using magnetic tweezers (MT) is a powerful method to probe the physical characteristics of single polymers. Typically, molecules are functionalized for specific attachment to a glass surface at one end and a micron-scale paramagnetic beads at the other. By applying an external magnetic field, multiple molecules can be stretched and twisted simultaneously without exposure to potentially damaging radiation. The majority of MT utilize moving permanent magnets to produce the forces on the beads (and the molecule under test). However, translating and rotating the permanent magnets may require expensive precision actuators, limits the rate at which force is changed, and may induce vibrations that disturb tether dynamics. Alternatively, the magnetic field can be produced through an electromagnet which allows much faster force modulation and eliminates motor-associated vibration. Here, we describe a low-cost quadrapolar electromagnetic tweezer design capable of manipulating DNA-tethered MyOne paramagnetic beads with forces of up to 20 pN. The solid-state nature of the generated B-field modulated along two axes is convenient for accessing the range of forces and torques relevant for studying the activity of DNA motor enzymes like polymerases and helicases. Our design specifically leverages technology available at an increasing number university maker spaces and student-run machine shops. Thus, our design is not only applicable to a wide range biophysical research questions, but also an accessible tool for undergraduate education., 7 pages, 7 figures

Loops are ubiquitous topological elements formed when proteins simultaneously bind to two noncontiguous DNA sites. While a loop-mediating protein may regulate initiation at a promoter, the presence of the protein at the other site may be an obstacle for RNA polymerases (RNAP) transcribing a different gene. To test whether a DNA loop alters the extent to which a protein blocks transcription, the lac repressor (LacI) was used. The outcome of in vitro transcription along templates containing two LacI operators separated by 400 bp in the presence of LacI concentrations that produced both looped and unlooped molecules was visualized with scanning force microscopy (SFM). An analysis of transcription elongation complexes, moving for 60 s at an average of 10 nt/s on unlooped DNA templates, revealed that they more often surpassed LacI bound to the lower affinity O2 operator than to the highest affinity Os operator. However, this difference was abrogated in looped DNA molecules where LacI became a strong roadblock independently of the affinity of the operator. Recordings of transcription elongation complexes, using magnetic tweezers, confirmed that they halted for several minutes upon encountering a LacI bound to a single operator. The average pause lifetime is compatible with RNAP waiting for LacI dissociation, however, the LacI open conformation visualized in the SFM images also suggests that LacI could straddle RNAP to let it pass. Independently of the mechanism by which RNAP bypasses the LacI roadblock, the data indicate that an obstacle with looped topology more effectively interferes with transcription.

This editorial highlights the pervasive role of DNA supercoiling in genomic structure and function and introduces the main themes of the reviews in this Special Issue.

Tethered Particle Motion (TPM) is a versatile in vitro technique for monitoring the conformations a linear macromolecule, such as DNA, can exhibit. The technique involves monitoring the diffusive motion of a particle anchored to a fixed point via the macromolecule of interest, which acts as a tether. In this chapter, we provide an overview of TPM, review the fundamental principles that determine the accuracy with which effective tether lengths can be used to distinguish different tether conformations, present software tools that assist in capturing and analyzing TPM data, and provide a protocol which uses TPM to characterize lac repressor-induced DNA looping. Critical to any TPM assay is the understanding of the timescale over which the diffusive motion of the particle must be observed to accurately distinguish tether conformations. Approximating the tether as a Hookean spring, we show how to estimate the diffusion timescale and discuss how it relates to the confidence with which tether conformations can be distinguished. Applying those estimates to a lac repressor titration assay, we describe how to perform a TPM experiment. We also provide graphically driven software which can be used to speed up data collection and analysis. Lastly, we detail how TPM data from the titration assay can be used to calculate relevant molecular descriptors such as the J factor for DNA looping and lac repressor-operator dissociation constants. While the included protocol is geared toward studying DNA looping, the technique, fundamental principles, and analytical methods are more general and can be adapted to a wide variety of molecular systems.

DNA is the carrier of genetic information and, as such, is at the center of most essential cellular processes. To regulate its physiological function, specific proteins and motor enzymes constantly change conformational states with well-controlled dynamics. Twenty-five years ago, Schafer, Gelles, Sheetz, and Landick employed the tethered particle motion (TPM) technique for the first time to study transcription by RNA polymerase at the single-molecule level. TPM has since then remained one of the simplest, most affordable, and yet incisive single-molecule techniques available. It is an in vitro technique which allows investigation of DNA–protein interactions that change the effective length of a DNA tether. In this chapter, we will describe a recent strategy to multiplex TPM which substantially increases the throughput of TPM experiments, as well as a simulation to estimate the time resolution of experiments, such as transcriptional elongation assays, in which lengthy time averaging of the signal is impossible due to continual change of the DNA tether length. These improvements allow efficient study of several DNA–protein systems, including transcriptionally active DNA–RNA polymerase I complexes and DNA–gyrase complexes.

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.

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.

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.

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.

Type II topoisomerases modify DNA supercoiling, and crystal structures suggest that they sharply bend DNA in the process. Bacterial gyrases are a class of type II topoisomerases that can introduce negative supercoiling by creating a wrap of DNA before strand passage. Isoforms of these essential enzymes were compared to reveal whether they can bend or wrap artificially stiffened DNA. Escherichia coli gyrase and human topoisomerase IIα were challenged with normal DNA or stiffer DNA produced by polymerase chain reaction reactions in which diaminopurine (DAP) replaced adenine deoxyribonucleotide triphosphates. On single DNA molecules twisted with magnetic tweezers to create plectonemes, the rates or pauses during relaxation of positive supercoils in DAP-substituted versus normal DNA were distinct for both enzymes. Gyrase struggled to bend or perhaps open a gap in DAP-substituted DNA, and segments of wider DAP DNA may have fit poorly into the N-gate of the human topoisomerase IIα. Pauses during processive activity on both types of DNA exhibited ATP dependence consistent with two pathways leading to the strand-passage-competent state with a bent gate segment and a transfer segment trapped by an ATP-loaded and latched N-gate. However, E. coli DNA gyrase essentially failed to negatively supercoil 35% stiffer DAP DNA.

DNA loops mediated by proteins that bind with different affinity to distant sites regulate multiple aspects of DNA metabolism, such as transcription, replication and recombination. Binding of accessory proteins and DNA supercoiling are two factors that affect the formation of DNA loops. Previous studies indicate that the heat unstable protein (HU) enhances the formation of short loops perhaps by changing the flexibility of DNA upon binding non-specifically. However, for loops long enough such that DNA stiffness is not limiting, the effect of HU on loop formation is not well understood. Negative supercoiling also enhances the formation of long loops. To better understand how binding affinity, loop length, HU and negative supercoiling affect looping, single molecule experiments were performed with the lac repressor protein (LacI) as a DNA looping protein. Using tethered particle motion, a titration of the formation of Lac repressor mediated-loop was carried out using three different DNA templates, OID-900-O1, OID-400-O1, and O1-400-O2 (Ostrong-loop size (bp)-Oweak). It showed that long loops are surprisingly efficient across a broad range of concentration. In similar titrations of LacI-induced looping as a function of HU concentration, HU greatly enhanced looping in 400 but not the longer 900 bp loops. Magnetic tweezers were used to investigate the effect of supercoiling on the formation of LacI-mediated DNA loops using 2 kbp DNA molecules with a centrally located O1-400-O2. Even slight tension in the magnetic tweezer interfered with looping, however, supercoiling compensated for increased tension. Positive supercoiling enhanced loop formation but less efficiently than negative which increased the looping probability from 0 to 100%. Thus the levels of accessory proteins and supercoiling, two dynamic parameters, enable modulation of regulatory loops the baseline probability of which is established by loop size and binding site affinities.

Quantitative analysis of single molecule experiments show that adding either of two natural polyamines, spermine or spermidine, produced more compact plectonemes in DNA in physiological concentrations of monovalent salt. They also promoted plectoneme formation at lower values of torsion in measurements of extension versus twist. Quantifying changes in the plectonemic DNA using some results from simple rod models suggested that exposure to polyamines reduced the radii and increased the densities of plectonemes. Thus, polyamines may limit the twist density by favoring writhe which maintains the B-form. Although polymerases may significantly stretch the double helix, denature DNA, and produce twist instead of writhe, natural polyamines stabilize base-pairing, limit twist to maintain the B-form, and promote supercoiling, which is conducive to replication and transcription and essential for DNA packaging.

Single-molecule experimentation has contributed significantly to our understanding of the mechanics of nucleoprotein complexes that regulate epigenetic switches. In this minireview, we will discuss the application of the tethered-particle motion technique, magnetic tweezers, and atomic force microscopy to (i) directly visualize and thermodynamically characterize DNA loops induced by the lac, gal, and lambda repressors and (ii) understand the mechanistic role of DNA-supercoiling and DNA-bending cofactors in both prokaryotic and eukaryotic systems.

Large, cooperative assemblies of proteins that wrap and/or loop genomic DNA may "epigenetically" shift configurational equilibria that determine developmental pathways. Such is the case of the lambda bacteriophage which may exhibit virulent (lytic) or quiescent (lysogenic) growth. The lysogenic state of lambda prophages is maintained by the lambda repressor (CI), which binds to tripartite operator sites in each of the O(L) and O(R) control regions located about 2.3 kbp apart on the phage genome and represses lytic promoters. Dodd and collaborators have suggested that an initial loop formed by interaction between CI bound at O(R) and O(L) provides the proper scaffold for additional CI binding to attenuate the P(RM) promoter and avoid over production of CI. Recently, the looping equilibrium as a function of CI concentration was measured using tethered particle motion analysis, but the oligomerization of CI in looped states could not be determined. Scanning force microscopy has now been used to probe these details directly. An equilibrium distribution of looped and unlooped molecules confined to a plane was found to be commensurate to that for tethered molecules in solution, and the occupancies of specific operator sites for several looped and unlooped conformations were determined. Some loops appeared to be sealed by oligomers of 6-8, most by oligomers of 10-12, and a few by oligomers of 14-16.

The nuclear factor-Y (NF-Y), a trimeric, CCAAT-binding transcriptional activator with histone-like subunits, was until recently considered a prototypical promoter transcription factor. However, recent in vivo chromatin immunoprecipitation assays associated with microarray methodologies (chromatin immunoprecipitation on chip experiments) have indicated that a large portion of target sites (40%–50%) are located outside of core promoters. We applied the tethered particle motion technique to the major histocompatibility complex class II enhancer-promoter region to characterize i), the progressive compaction of DNA due to increasing concentrations of NF-Y, ii), the role of specific subunits and domains of NF-Y in the process, and iii), the interplay between NF-Y and the regulatory factor-X, which cooperatively binds to the X-box adjacent to the CCAAT box. Our study shows that NF-Y has histone-like activity, since it binds DNA nonspecifically with high affinity to compact it. This activity, which depends on the presence of all trimer subunits and of their glutamine-rich domains, seems to be attenuated by the transcriptional cofactor regulatory factor-X. Most importantly NF-Y-induced DNA compaction may facilitate promoter-enhancer interactions, which are known to be critical for expression regulation.