DNA Twist Research Articles

Topological Polymorphism of Two-Start Nucleosome Fibers: A Stereochemical Analysis

The spatial organization of nucleosomes in 30-nm fibers remains unknown in detail. To address this issue, we analyzed all stereochemically possible configurations of two-start nucleosome fibers with short DNA linkers L = 13 - 37 bp (nucleosome repeat length NRL = 160 - 184 bp). Four superhelical parameters – inclination of nucleosomes, twist, rise and diameter – uniquely describe a uniform symmetric fiber. The energy of a fiber is defined as the sum of four terms: elastic energy of the linker DNA, steric repulsion, electrostatics and a phenomenological (H4 tail - acidic patch) interaction between two stacked nucleosomes. By optimizing the fiber energy with respect to the superhelical parameters, we found two types of topological transition in fibers (associated with the change in inclination angle): one caused by an abrupt 360° change in the linker DNA twisting, and another caused by over-crossing of the linkers. (The first transition is characterized by change in the DNA linking number, ΔLk = 1, and the second one by ΔLk = 2.) To the best of our knowledge, this topological polymorphism of the two-start fibers was not reported in the computations published earlier. Importantly, the optimal configurations of the fibers with linkers L = 10n and 10n+5 bp are topologically different. Our results are consistent with experimental observations, such as the inclination 60-70° (the angle between the nucleosomal disks and the fiber axis), helical rise, diameter and left-handedness of the fibers. In addition, we make several testable predictions, among them existence of different degree of DNA supercoiling in the fibers with L = 10n and 10n+5 bp, different stiffness of the two types of fibers, and a correlation between the local NRL and the level of transcription in different parts of the yeast genome.

Changing Chromatin Fiber Conformation by Nucleosome Repositioning

Chromatin conformation is dynamic and heterogeneous with respect to nucleosome positions, which can be changed by chromatin remodeling complexes in the cell. These molecular machines hydrolyze ATP to translocate or evict nucleosomes, and establish loci with regularly and more irregularly spaced nucleosomes as well as nucleosome-depleted regions. The impact of nucleosome repositioning on the three-dimensional chromatin structure is only poorly understood. Here, we address this issue by using a coarse-grained computer model of arrays of 101 nucleosomes considering several chromatin fiber models with and without linker histones, respectively. We investigated the folding of the chain in dependence of the position of the central nucleosome by changing the length of the adjacent linker DNA in basepair steps. We found in our simulations that these translocations had a strong effect on the shape and properties of chromatin fibers: i), Fiber curvature and flexibility at the center were largely increased and long-range contacts between distant nucleosomes on the chain were promoted. ii), The highest destabilization of the fiber conformation occurred for a nucleosome shifted by two basepairs from regular spacing, whereas effects of linker DNA changes of ∼10 bp in phase with the helical twist of DNA were minimal. iii), A fiber conformation can stabilize a regular spacing of nucleosomes inasmuch as favorable stacking interactions between nucleosomes are facilitated. This can oppose nucleosome translocations and increase the energetic costs for chromatin remodeling. Our computational modeling framework makes it possible to describe the conformational heterogeneity of chromatin in terms of nucleosome positions, and thus advances theoretical models toward a better understanding of how genome compaction and access are regulated within the cell.

Search by proteins for their DNA target site: 1. The effect of DNA conformation on protein sliding.

The recognition of DNA-binding proteins (DBPs) to their specific site often precedes by a search technique in which proteins slide, hop along the DNA contour or perform inter-segment transfer and 3D diffusion to dissociate and re-associate to distant DNA sites. In this study, we demonstrated that the strength and nature of the non-specific electrostatic interactions, which govern the search dynamics of DBPs, are strongly correlated with the conformation of the DNA. We tuned two structural parameters, namely curvature and the extent of helical twisting in circular DNA. These two factors are mutually independent of each other and can modulate the electrostatic potential through changing the geometry of the circular DNA conformation. The search dynamics for DBPs on circular DNA is therefore markedly different compared with linear B-DNA. Our results suggest that, for a given DBP, the rotation-coupled sliding dynamics is precluded in highly curved DNA (as well as for over-twisted DNA) because of the large electrostatic energy barrier between the inside and outside of the DNA molecule. Under such circumstances, proteins prefer to hop in order to explore interior DNA sites. The change in the balance between sliding and hopping propensities as a function of DNA curvature or twisting may result in different search efficiency and speed.

Torsional behavior of chromatin is modulated by rotational phasing of nucleosomes.

Torsionally stressed DNA plays a critical role in genome organization and regulation. While the effects of torsional stresses on naked DNA have been well studied, little is known about how these stresses propagate within chromatin and affect its organization. Here we investigate the torsional behavior of nucleosome arrays by means of Brownian dynamics simulations of a coarse-grained model of chromatin. Our simulations reveal a strong dependence of the torsional response on the rotational phase angle Ψ0 between adjacent nucleosomes. Extreme values of Ψ0 lead to asymmetric, bell-shaped extension-rotation profiles with sharp maxima shifted toward positive or negative rotations, depending on the sign of Ψ0, and to fast, irregular propagation of DNA twist. In contrast, moderate Ψ0 yield more symmetric profiles with broad maxima and slow, uniform propagation of twist. The observed behavior is shown to arise from an interplay between nucleosomal transitions into states with crossed and open linker DNAs and global supercoiling of arrays into left- and right-handed coils, where Ψ0 serves to modulate the energy landscape of nucleosomal states. Our results also explain the torsional resilience of chromatin, reconcile differences between experimentally measured extension-rotation profiles, and suggest a role of torsional stresses in regulating chromatin assembly and organization.

Torque correlation length and stochastic twist dynamics of DNA.

We introduce a short correlation length for torque in twisting-stiff biomolecules, which is necessary for the physical property that torque fluctuations be finite in amplitude. We develop a nonequilibrium theory of dynamics of DNA twisting which predicts two crossover time scales for temporal torque correlations in single-molecule experiments. Bending fluctuations can be included, and at linear order we find that they do not affect the twist dynamics. However, twist fluctuations affect bending, and we predict the spatial inhomogeneity of twist, torque, and buckling arising in nonequilibrium "rotor-bead" experiments.

Gold rotor bead tracking for high-speed measurements of DNA twist, torque and extension

Simultaneous measurements of DNA twist and extension have been used to measure physical properties of the double helix and to characterize structural dynamics and mechanochemistry in nucleoprotein complexes. However, the spatiotemporal resolution of twist measurements has been limited by the use of angular probes with large rotational drags, preventing the detection of short-lived intermediates or small angular steps. Here we introduce AuRBT, demonstrating a >100X improvement in time resolution over previous techniques. AuRBT employs gold nanoparticles as bright low-drag rotational and extensional probes, relying on instrumentation that combines magnetic tweezers with objective-side evanescent darkfield microscopy. In an initial application to molecular motor mechanism, we have examined the high-speed structural dynamics of DNA gyrase, revealing an unanticipated transient intermediate. AuRBT also enables direct measurements of DNA torque with >50X shorter integration times than previous techniques; here we demonstrate high-resolution torque spectroscopy by mapping the conformational landscape of a Z-forming DNA sequence.

Novel Coarse-Grained Model for Molecular Dynamics Simulations of DNA

We have developed and efficient, sequence-specific coarse-grained model to simulate the conformational motion of large double stranded DNA in molecular dynamics simulations. The DNA backbone is modeled by beads with DNA bases represented by sets of three beads, and bead positions are used to calculate the shift, slide, rise, roll, twist and tilt conformational step parameters of DNA. With these parameters, elastic deformation energies are calculated following Olson's harmonic model (Olson et. al., Proc. Natl. Acad. Sci. USA 95, 11163 (1998)). Specifics of the model and sample simulations will be discussed.

Torsional Behavior of Nucleosome Arrays

Genomic DNA is subjected to strong torsional stresses during processes like transcription and replication. Emerging evidence suggests that such torsional stresses might serve critical functions in the cell nucleus. Most experimental and theoretical work so far has focused on understanding the supercoiling of the naked DNA, but little is known about how torsional stresses are stored, propagated, and relieved within the chromatin fiber. Here we investigate the torsional behavior of nucleosome arrays by using Brownian dynamics simulations of a coarse-grained model of chromatin fiber. By rotating one end of the array in a step-wise manner while keeping the other end fixed, we measure the equilibrium extension of the array as a function of the applied twist. The measured twist-extension profiles display an asymmetry shape with a shifted maximal extension. The magnitude and sign of the observed shift depends strongly on the internucleosomal “phase” angle resulting from the helical twist of the linker DNA. Moreover, moderate values of the phase angle lead to broad plateau at the maxima in the twist-extension plots while extreme values of the angle lead to sharper maxima. By tracing different nucleosome states in the rotations, we show that the plateau results from a conformational transition between open and negatively crossed states of individual nucleosomes with a minimal twisting energy cost, explaining high torsional resilience of the array. Indeed, the torsional rigidity of the array extracted from twisting energies is significantly smaller than that of naked DNA. Finally, we construct the kymograph of rotational autocorrelation function for each segment of the array, which clearly visualizes the effect of phase angle on the propagation of twist. This work provides one of the first detailed pictures of supercoiled chromatin and how eukaryotic DNA might relieve its torsional stresses through conformational transitions in the nucleosome.

Mechanical Strain Generated by RNA Polymerase during Transcription Initiation can Drive Structural Changes in DNA Topology that Relieve Repression

DNA in cells is often topologically closed, and in some cases, tightly looped by proteins typically associated with transcriptional repression. During transcription initiation, RNA polymerase (RNAP) is challenged to open duplex DNA, wherein RNAP generates torque that consequently overtwists DNA flanking the melted promoter. As initiation proceeds, the transcription bubble expands as RNAP synthesizes downstream RNA while maintaining upstream DNA contacts. We hypothesized that the mechanical strain imparted to DNA during initiation would repress transcription from DNA templates that restrict the relief of torsional stress. To test our hypothesis, we constructed circular DNA templates that are 100 to 108 bp in size (i.e., each initially twisted to various degrees) and quantified transcription initiation by the bacteriophage T7 RNAP. We find that transcriptional repression during initiation is dependent on the sign and magnitude of initial twist within the DNA templates. Surprisingly, however, we observe that for the most overtwisted templates, repression is relieved at positions beyond the promoter that are dependent on the initial DNA twist. To interpret these results, we used elastic rod and molecular dynamics simulations to predict the structures of both the RNAP and the circular DNA template during initiation. Our modeling studies confirm that RNAP is capable of overtwisting the circular DNA template to the point of buckling from a planar into a supercoiled conformation. Further analysis reveals that initial DNA twist determines at what position along the template supercoiling will occur during initiation and that adopting a supercoiled structure substantially relieves the torque encountered by the RNAP. Our results demonstrate that repression of RNAP during initiation is determined not only by the initial mechanics of the DNA template, but also by the torque generated by RNAP itself.

Mechanical Properties of DNA-Like Polymers

This work continues to probe the mystery of what balance of forces creates the extraordinary mechanical stiffness of DNA to bending and twisting. In particular, we have been trying to understand the extent to which the charge of DNA contributes to stiffness through electrostatic stretching forces. Remarkably, neither theory nor experiment has resolved this issue over six decades. We have approached the problem by making a family of double-helical DNA-like polymers in which one of the four normal bases is replaced with an anionic, cationic, or neutral analog that alters canonical stacking, charge, or both. The resulting DNA-like polymers were characterized in a variety of ways, including determining bend stiffness using cyclization kinetics and atomic force microscopy experiments. Recent theory predicts that the variants with different charge densities should have dramatically altered bending rigidity. Remarkably, we show that these DNA-like polymers have bend stiffness within the range of reported values for sequence-dependent variation of the natural DNA bases. On the other hand, DNA twist flexibility was strikingly different among the polymers. These results suggest that electrostatic stretching forces do not contribute measurably to DNA bend stiffness, showing the inadequacy of recent theory and the need for new concepts.

Elastic Twist Dynamics of DNA

Single-molecule DNA experiments have revealed a number of observations about the mechanical properties of DNA, but little is known about the dynamics of twist and bend in these experiments. To understand these experiments, we develop and solve a stochastic and elastic model for twist dynamics in a DNA molecule that is driven at one end and dissipates torsional energy through viscous drag at the other end. To ensure that torque fluctuations remain finite in our model, we include a term depending on the gradient-squared of twist strain. We find that the boundary conditions of our system quantize the spectrum of DNA twisting modes and modulate the dynamics in a striking way. To see this, we derive a nonequilibrium fluctuation relation for twist and construct the spatiotemporal correlation function. We find that a novel elastic correlation length controls spatial correlations between twist fluctuations. In the middle of a long DNA molecule, twist correlations initially decay as t−1/4 and crossover to t−1/2 decay at longer times. In contrast, in a shorter molecule, correlations decay exponentially with a decay time that depends on the magnitude of the viscous drag. For nearly straight DNA molecules, twist dynamics are unaffected by bending fluctuations at linear order, even though writhing dynamics are modulated by twist at this order. Our model for twist dynamics suggests several new single-molecule experiments, which could shed further light on DNA mechanics and dynamics.

Time-dependent bending rigidity and helical twist of DNA by rearrangement of bound HU protein

HU is a protein that plays a role in various bacterial processes including compaction, transcription and replication of the genome. Here, we use atomic force microscopy to study the effect of HU on the stiffness and supercoiling of double-stranded DNA. First, we measured the persistence length, height profile, contour length and bending angle distribution of the DNA–HU complex after different incubation times of HU with linear DNA. We found that the persistence and contour length depend on the incubation time. At high concentrations of HU, DNA molecules first become stiff with a larger value of the persistence length. The persistence length then decreases over time and the molecules regain the flexibility of bare DNA after ∼2 h. Concurrently, the contour length shows a slight increase. Second, we measured the change in topology of closed circular relaxed DNA following binding of HU. Here, we observed that HU induces supercoiling over a similar time span as the measured change in persistence length. Our observations can be rationalized in terms of the formation of a nucleoprotein filament followed by a structural rearrangement of the bound HU on DNA. The rearrangement results in a change in topology, an increase in bending flexibility and an increase in contour length through a decrease in helical pitch of the duplex.

Structural and torsional properties of the RAD51-dsDNA nucleoprotein filament

Human RAD51 is a key protein in the repair of DNA by homologous recombination. Its assembly onto DNA, which induces changes in DNA structure, results in the formation of a nucleoprotein filament that forms the basis of strand exchange. Here, we determine the structural and mechanical properties of RAD51-dsDNA filaments. Our measurements use two recently developed magnetic tweezers assays, freely orbiting magnetic tweezers and magnetic torque tweezers, designed to measure the twist and torque of individual molecules. By directly monitoring changes in DNA twist on RAD51 binding, we determine the unwinding angle per RAD51 monomer to be 45°, in quantitative agreement with that of its bacterial homolog, RecA. Measurements of the torque that is built up when RAD51-dsDNA filaments are twisted show that under conditions that suppress ATP hydrolysis the torsional persistence length of the RAD51-dsDNA filament exceeds that of its RecA counterpart by a factor of three. Examination of the filament’s torsional stiffness for different combinations of divalent ions and nucleotide cofactors reveals that the Ca2+ ion, apart from suppressing ATPase activity, plays a key role in increasing the torsional stiffness of the filament. These quantitative measurements of RAD51-imposed DNA distortions and accumulated mechanical stress suggest a finely tuned interplay between chemical and mechanical interactions within the RAD51 nucleoprotein filament.

Chiral Discrimination and Writhe-dependent Relaxation Mechanism of Human Topoisomerase IIα

Human topoisomerase IIα unlinks catenated chromosomes and preferentially relaxes positive supercoils. Supercoil chirality, twist density, and tension determine topoisomerase IIα relaxation rate and processivity. Strand passage rate is determined by the efficiency of transfer segment capture that is modulated by the topoisomerase C-terminal domains. Single-molecule measurements reveal the mechanism of chiral discrimination and tension dependence of supercoil relaxation by human topoisomerase IIα. Type IIA topoisomerases (Topo IIA) are essential enzymes that relax DNA supercoils and remove links joining replicated chromosomes. Human topoisomerase IIα (htopo IIα), one of two human isoforms, preferentially relaxes positive supercoils, a feature shared with Escherichia coli topoisomerase IV (Topo IV). The mechanistic basis of this chiral discrimination remains unresolved. To address this important issue, we measured the relaxation of individual supercoiled and "braided" DNA molecules by htopo IIα using a magnetic tweezers-based single-molecule assay. Our study confirmed the chiral discrimination activity of htopo IIα and revealed that the strand passage rate depends on DNA twist, tension on the DNA, and the C-terminal domain (CTD). Similar to Topo IV, chiral discrimination by htopo IIα results from chiral interactions of the CTDs with DNA writhe. In contrast to Topo IV, however, these interactions lead to chiral differences in relaxation rate rather than processivity. Increasing tension or twist disrupts the CTD-DNA interactions with a subsequent loss of chiral discrimination. Together, these results suggest that transfer segment (T-segment) capture is the rate-limiting step in the strand passage cycle. We propose a model for T-segment capture that provides a mechanistic basis for chiral discrimination and provides a coherent explanation for the effects of DNA twist and tension on eukaryotic type IIA topoisomerases.

Life's ratchet: how molecular machines extract order from chaos

Life is an enduring mystery. Yet, science tells us that living beings are merely sophisticated structures of lifeless molecules. If this view is correct, where do the seemingly purposeful motions of cells and organisms originate? In Life's Ratchet, physicist Peter M. Hoffmann locates the answer to this age-old question at the nanoscale. Below the calm, ordered exterior of a living organism lies microscopic chaos, or what Hoffmann calls the molecular storm--specialized molecules immersed in a whirlwind of colliding water molecules. Our cells are filled with molecular machines, which, like tiny ratchets, transform random motion into ordered activity, and create the purpose that is the hallmark of life. Tiny electrical motors turn electrical voltage into motion, nanoscale factories custom-build other molecular machines, and mechanical machines twist, untwist, separate and package strands of DNA. The cell is like a city--an unfathomable, complex collection of molecular workers working together to create something greater than themselves. Life, Hoffman argues, emerges from the random motions of atoms filtered through these sophisticated structures of our evolved machinery. We are agglomerations of interacting nanoscale machines more amazing than anything in science fiction. Rather than relying on some mysterious force to drive them--as people believed for centuries--life's ratchets harness instead the second law of thermodynamics and the disorder of the molecular storm. Grounded in Hoffmann's own cutting-edge research, Life's Ratchet reveals the incredible findings of modern nanotechnology to tell the story of how the noisy world of atoms gives rise to life itself.

A solution to release twisted DNA during chromosome replication by coupled DNA polymerases

Chromosomal replication machines contain coupled DNA polymerases that simultaneously replicate the leading and lagging strands1. However, coupled replication presents a largely unrecognized topological problem. Since DNA polymerase must travel a helical path during synthesis, the physical connection between leading and lagging strand polymerases causes the daughter strands to entwine, or produces extensive buildup of negative supercoils in the newly synthesized DNA2–4. How DNA polymerases maintain their connection during coupled replication despite these topological challenges is a mystery. Here, we examine the dynamics of the E. coli replisome, by ensemble and single-molecule methods that may solve this topological problem independent of topoisomerases. We find that the lagging strand polymerase frequently releases from an Okazaki fragment before completion, leaving single-strand gaps behind. Dissociation of the polymerase does not result in loss from the replisome due to its contact with the leading-strand polymerase. This behavior, referred to as “signal release”, had been thought to require a protein, possibly primase, to pry polymerase from incompletely extended DNA fragments5–7. However, we observe that signal release is independent of primase and does not appear to require a protein trigger at all. Instead, the lagging-strand polymerase is simply less processive in the context of a replisome. Interestingly, when the lagging-strand polymerase is supplied with primed DNA in trans, uncoupling it from the fork, high processivity is restored. Hence, we propose that coupled polymerases introduce topological changes, possibly by accumulation of superhelical tension in the newly synthesized DNA, that cause lower processivity and transient lagging-strand polymerase dissociation from DNA.

On the Origin of Thermal Untwisting of DNA

In aqueous solutions, the helical twist of DNA decreases with temperature. This phenomenon was noticed and studied experimentally several decades ago, but its physical origin remains elusive. The present paper shows that the thermal untwisting can be predicted from the specific properties of the torsional elasticity of the double helix revealed in recent computational studies. The temperature coefficient of untwisting estimated using coarse-grained models fitted to all-atom MD data accounts for the experimental results nearly quantitatively. The agreement is further improved with the computed torsional rigidity scaled to remove the discrepancy from experiment. The results confirm that the torsional rigidity of DNA is strongly anharmonic. They indicate that for random DNA, its value grows with small twisting and decreases with untwisting.

Probing Rad51-DNA Interactions by Changing DNA Twist

In eukaryotes, Rad51 protein is responsible for the recombinational repair of double-strand DNA breaks. Rad51 monomers cooperatively assemble on exonuclease-processed broken ends forming helical nucleo-protein filaments that can pair with homologous regions of sister chromatids. Homologous pairing allows the broken ends to be reunited in a complex but error-free repair process. Rad51 protein has ATPase activity but its role is poorly understood, as homologous pairing is independent of adenosine triphosphate (ATP) hydrolysis. Here we use magnetic tweezers and electron microscopy to investigate how changes of DNA twist affect the structure of Rad51-DNA complexes and how ATP hydrolysis participates in this process. We show that Rad51 protein can bind to double-stranded DNA in two different modes depending on the enforced DNA twist. The stretching mode is observed when DNA is unwound towards a helical repeat of 18.6bp/turn, whereas a non-stretching mode is observed when DNA molecules are not permitted to change their native helical repeat. We also show that the two forms of complexes are interconvertible and that by enforcing changes of DNA twist one can induce transitions between the two forms. Our observations permit a better understanding of the role of ATP hydrolysis in Rad51-mediated homologous pairing and strand exchange.

64 Hoogsteen or not Hoogsteen? Iodine-125 radioprobing of the p53-induced DNA deformations

Radioprobing is suitable for tracing the DNA and RNA trajectories in nucleoprotein complexes in solution. The method is based on the analysis of the single-strand breaks produced by decay of iodine-125 incorporated in the C5 position of cytosine (Karamychev et al., 1999, 2012). Here, we used radioprobing to study the conformation of DNA in complex with the DNA binding domain (DBD) of the tumor-suppressor protein p53. Two recently crystallized DNA-p53 DBD complexes have different conformations of the CATG motifs: one with the Hoogsteen A:T pairs (Kitayner et al., 2010) and the other with the Watson–Crick pairs (Chen et al., 2010). The two complexes differ in the sequence of the central YYY|RRR junction: the first one has the C|G step and the second has the T|A step. Thus, it is interesting to apply the radioprobing method to the two DNA sequences used in crystallography to see if the local changes (T|A to C|G) in the center of the p53 response element would produce significant distortions in the CATG motifs. To this aim, the iodine-containing cytosine was incorporated in the duplexes containing p53-binding sites, in one of the two CATG motifs and the frequencies of DNA breaks were analyzed. Frequencies of breaks are negatively correlated with the iodine–sugar distances, thus, one can evaluate the changes in these distances upon DNA binding to a protein. The radioprobing distances obtained for both DNA sequences proved to be consistent with the Watson–Crick structure observed by Chen et al. (2010). We did not find any evidence of the Hoogsteen A:T base pair formation in the DNA-p53 DBD complexes in solution using our radioprobing method. The most significant changes in the break frequency distributions were detected in the central segment of the p53 binding site, YYY|RRR, which are consistent with an increase in DNA twisting in this region and local DNA bending and sliding (Nagaich et al., 1999). We interpret these p53-induced DNA deformations in the context of p53 binding to nucleosomal DNA (Sahu et al., 2010).

Transcription on Tangled DNA

Bending and torsional deformations of DNA often occur in vivo and are important for various biological functions in the cell. In particular, DNA supercoiling (a combination of bending and torsional stresses within DNA) plays a central role in regulating gene expression. Recent experimental studies of short circular DNA fragments suggest that combined DNA torsional and bending stresses strongly impact transcription by RNA polymerase (RNAP). However, despite phenomenological observations establishing a relationship between DNA supercoiling and transcription kinetics, many details of the structure-function relationship between RNAP and DNA template mechanics remain unclear. We aim to understand how supercoiled DNA affects the structure of DNA-bound RNA polymerase, with the goal of specifically identifying protein domains that are sensitive to mechanically stressed DNA templates. Massively parallel molecular dynamics simulations are performed to describe the interaction of bacteriophage T7-RNAP with three DNA minicircles possessing qualitatively distinct states of torsional stress: underwound, overwound, and relaxed. The simulations reveal the topologies of the minicircles in complex with T7-RNAP and the structural details of bent and twisted protein-bound DNA. We observe remarkable differences between the conformations of T7-RNAP in complex with undertwisted, overtwisted and relaxed minicircle templates. In particular, on overtwisted DNA, T7-RNAP is capable of transitioning from the classical elongation complex structure into a stable intermediate that resembles the structure immediately preceding elongation. This finding suggests a structural mechanism by which transcription elongation may be hindered on bent and overtwisted DNA templates.