Transcription Bubble Research Articles

Structure and mechanism of the plant RNA polymerase V

In addition to the conserved RNA polymerases I to III (Pols I to III) in eukaryotes, two atypical polymerases, Pols IV and V, specifically produce noncoding RNA in the RNA-directed DNA methylation pathway in plants. Here, we report on the structures of cauliflower Pol V in the free and elongation conformations. A conserved tyrosine residue of NRPE2 stacks with a double-stranded DNA branch of the transcription bubble to potentially attenuate elongation by inducing transcription stalling. The nontemplate DNA strand is captured by NRPE2 to enhance backtracking, thereby increasing 3'-5' cleavage, which likely underpins Pol V's high fidelity. The structures also illuminate the mechanism of Pol V transcription stalling and enhanced backtracking, which may be important for Pol V's retention on chromatin to serve its function in tethering downstream factors for RNA-directed DNA methylation.

Allosteric mechanism of transcription inhibition by NusG-dependent pausing of RNA polymerase.

NusG is a transcription elongation factor that stimulates transcription pausing in Gram+ bacteria including B. subtilis by sequence-specific interaction with a conserved pause-inducing -11TTNTTT-6 motif found in the non-template DNA (ntDNA) strand within the transcription bubble. To reveal the structural basis of NusG-dependent pausing, we determined a cryo-EM structure of a paused transcription complex (PTC)containing RNA polymerase (RNAP), NusG, and the TTNTTT motif in the ntDNA strand. The interaction of NusG with the ntDNA strand rearranges the transcription bubble by positioning three consecutive T residues in a cleft between NusG and the β-lobe domain of RNAP. We revealed that the RNAP swivel module rotation (swiveling), which widens (swiveled state) and narrows (non-swiveled state) a cleft between NusG and the β-lobe, is an intrinsic motion of RNAP and is directly linked to trigger loop (TL) folding, an essential conformational change of all cellular RNAPs for the RNA synthesis reaction. We also determined cryo-EM structures of RNAP escaping from the paused transcription state. These structures revealed the NusG-dependent pausing mechanism by which NusG-ntDNA interaction inhibits the transition from swiveled to non-swiveled states, thereby preventing TL folding and RNA synthesis allosterically. This motion is also reduced by the formation of an RNA hairpin within the RNA exit channel. Thus, the pause half-life can be modulated by the strength of the NusG-ntDNA interaction and/or the stability of the RNA hairpin. NusG residues that interact with the TTNTTT motif are widely conserved in bacteria, suggesting that NusG-dependent pausing is widespread.

Structural basis for intrinsic transcription termination.

Efficient and accurate termination is required for gene transcription in all living organisms1,2. Cellular RNA polymerases in both bacteria and eukaryotes can terminate their transcription through a factor-independent termination pathway3,4-called intrinsic termination transcription in bacteria-in which RNA polymerase recognizes terminator sequences, stops nucleotide addition and releases nascent RNA spontaneously. Here we report a set of single-particle cryo-electron microscopy structures of Escherichia coli transcription intrinsic termination complexes representing key intermediate states of the event. The structures show how RNA polymerase pauses at terminator sequences, how the terminator RNA hairpin folds inside RNA polymerase, and how RNA polymerase rewinds the transcription bubble to release RNA and then DNA. These macromolecular snapshots define a structural mechanism for bacterial intrinsic termination and a pathway for RNA release and DNA collapse that isrelevant for factor-independent termination by all RNA polymerases.

Rotation manipulation of single-molecule magnetic trapping and gene transcription regulation dynamics

Gene transcription regulation is a key step for gene expression in all organisms and responsible for the transmission of genetic information and genome integrity. As one of the most important mechanisms in gene transcription, an RNA polymerase (RNAP) specifically interacts with and unwinds genome DNA to form a transcription bubble where a nascent RNA transcript is polymerized, taking one of the unwound DNA strands as its template. The RNAP translocates along the DNA to transcribe the whole gene by carrying the transcription bubble. In such a way, an RNAP completes its biological task of gene expression by physically acting as a molecular machinery. Thus, an RNAP molecule can be considered as a research object for physicists who are willing to uncover the mechanisms of life processes in a physical view. To achieve this, single-molecule method has been invented and used widely. As one of these methods, single-molecule magnetic trapping manipulates biological molecules by applying extension force or torque to the magnetic beads tethered through biological molecule to pre-coated glass surfaces by manipulating the position or rotation of a pair of magnets. A linear DNA molecule can be manipulated in such a way to generate plectonemes, i.e. DNA supercoils, under an extension force of 0.3 pN (1 pN = 10<sup>–12</sup> N), possessing the feature that the number of unwound base pairs of a supercoiled DNA can be observed by the changes in the number of supercoils reflected by the DNA extension changes. Thus, the DNA unwound by RNAP, i.e. the transcription bubble, during transcription can be observed in this way. By monitoring the kinetics of the transcription bubble in real time, this method thus allows single-molecule detection with single-base resolution and a high-throughput data collection fashion in the kinetic studies of transcription. Owing to the advantages of the manipulation of DNA supercoils with single-molecule magnetic trapping, one can mimic the mechanistic feature of DNAs in vivo and characterize the kinetics of transcription under such conditions. This method can also be combined with single-molecule fluorescence method which can be applied to studying the mechanism of transcription regulation while monitoring the behaviors of fluorescently labeled biological molecules that interact with functional RNAP molecules, providing examples for studying the mechanisms of transcription regulations in more complex systems.

On the dimensionless model of the transcription bubble dynamics

<abstract> <p>The dynamics of transcription bubbles is modeled using a system of nonlinear differential equations, the one-soliton solutions of which (kinks), are interpreted as a mathematical images of transcription bubbles. These equations contain a lot of DNA dynamic parameters, including the moments of inertia of nitrous bases, distances between base pairs, distances from the centers of mass of bases to sugar-phosphate chains, rigidity of the sugar-phosphate backbone, and interactions between bases within pairs. However, estimates of the parameter values are often difficult, and it is not convenient or simple to operate with such multi-parameter systems. One of the ways to reduce the number of the DNA dynamic parameters is to transform the model equations to a dimensionless form. In this work, we construct a dimensionless DNA model and apply it to study transcription bubbles dynamics. We show that transformation to a dimensionless form really leads to a decrease in the number of the model parameters and really simplifies the analysis of model equations and their solutions.</p> </abstract>

Thermodynamic model of bacterial transcription.

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.

Oligonucleotide-Recognizing Topoisomerase Inhibitors (OTIs): Precision Gene Editors for Neurodegenerative Diseases?

Topoisomerases are essential enzymes that recognize and modify the topology of DNA to allow DNA replication and transcription to take place. Topoisomerases are divided into type I topoisomerases, that cleave one DNA strand to modify DNA topology, and type II, that cleave both DNA strands. Topoisomerases normally rapidly religate cleaved-DNA once the topology has been modified. Topoisomerases do not recognize specific DNA sequences, but actively cleave positively supercoiled DNA ahead of transcription bubbles or replication forks, and negative supercoils (or precatenanes) behind, thus allowing the unwinding of the DNA-helix to proceed (during both transcription and replication). Drugs that stabilize DNA-cleavage complexes with topoisomerases produce cytotoxic DNA damage and kill fast-dividing cells; they are widely used in cancer chemotherapy. Oligonucleotide-recognizing topoisomerase inhibitors (OTIs) have given drugs that stabilize DNA-cleavage complexes specificity by linking them to either: (i) DNA duplex recognizing triplex forming oligonucleotide (TFO-OTIs) or DNA duplex recognizing pyrrole-imidazole-polyamides (PIP-OTIs) (ii) or by conventional Watson–Crick base pairing (WC-OTIs). This converts compounds from indiscriminate DNA-damaging drugs to highly specific targeted DNA-cleaving OTIs. Herein we propose simple strategies to enable DNA-duplex strand invasion of WC-OTIs giving strand-invading SI-OTIs. This will make SI-OTIs similar to the guide RNAs of CRISPR/Cas9 nuclease bacterial immune systems. However, an important difference between OTIs and CRISPR/Cas9, is that OTIs do not require the introduction of foreign proteins into cells. Recent successful oligonucleotide therapeutics for neurodegenerative diseases suggest that OTIs can be developed to be highly specific gene editing agents for DNA lesions that cause neurodegenerative diseases.

The human RNA polymerase I structure reveals an HMG-like docking domain specific to metazoans.

Transcription of the ribosomal RNA precursor by RNA polymerase (Pol) I is a major determinant of cellular growth, and dysregulation is observed in many cancer types. Here, we present the purification of human Pol I from cells carrying a genomic GFP fusion on the largest subunit allowing the structural and functional analysis of the enzyme across species. In contrast to yeast, human Pol I carries a single-subunit stalk, and in vitro transcription indicates a reduced proofreading activity. Determination of the human Pol I cryo-EM reconstruction in a close-to-native state rationalizes the effects of disease-associated mutations and uncovers an additional domain that is built into the sequence of Pol I subunit RPA1. This "dock II" domain resembles a truncated HMG box incapable of DNA binding which may serve as a downstream transcription factor-binding platform in metazoans. Biochemical analysis, in situ modelling, and ChIP data indicate that Topoisomerase 2a can be recruited to Pol I via the domain and cooperates with the HMG box domain-containing factor UBF. These adaptations of the metazoan Pol I transcription system may allow efficient release of positive DNA supercoils accumulating downstream of the transcription bubble.

Znhit1 controls meiotic initiation in male germ cells by coordinating with Stra8 to activate meiotic gene expression.

The switch from mitosis to meiosis ensures the successive formation of gametes. However, it remains unclear how meiotic initiation occurs within the context of chromatin. Recent studies have shown that zinc finger HIT-type containing 1 (Znhit1), a subunit of the SRCAP chromatin remodeling complex, plays essential roles in modulating the chromatin structure. Herein, we report that the germline-conditional deletion of Znhit1 in male mice specifically blocks meiotic initiation. We show that Znhit1 is required for meiotic prophase events, including synapsis, DNA double-strand break formation, and meiotic DNA replication. Mechanistically, Znhit1 controls the histone variant H2A.Z deposition, which facilitates the expression of meiotic genes, such as Meiosin, but not the expression of Stra8. Interestingly, Znhit1 deficiency disrupts the transcription bubbles of meiotic genes. Thus, our findings identify the essential role of Znhit1-dependent H2A.Z deposition in allowing activation of meiotic gene expression, thereby controlling the initiation of meiosis.

A thermodynamic model of bacterial transcription

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 observed in high-resolution optical tweezers measurements of transcription, in terms of pause sites, duration, and mechanisms.

DNA opening during transcription initiation by human RNA polymerase II in atomic details

RNA polymerase II (RNAPII) is a macro-molecular complex that synthesizes RNA by reading the DNA code, a process called transcription. During the initiation step of transcription, RNAPII opens double-stranded DNA in order to read the DNA code. Since formation of the DNA transcription bubble remains poorly understood, we used molecular dynamics simulations to provide atomic-level insights into this crucial step of transcription. Here, by steering the simulations with a combination of (i) guided DNA rotation and (ii) path collective variables, we obtained for the first time continuous atomic trajectories of the complete DNA opening process.

Integrated genome and transcriptome analyses reveal the mechanism of genome instability in ataxia with oculomotor apraxia 2

Mutations in the SETX gene, which encodes Senataxin, are associated with the progressive neurodegenerative diseases ataxia with oculomotor apraxia 2 (AOA2) and amyotrophic lateral sclerosis 4 (ALS4). To identify the causal defect in AOA2, patient-derived cells and SETX knockouts (human and mouse) were analyzed using integrated genomic and transcriptomic approaches. A genome-wide increase in chromosome instability (gains and losses) within genes and at chromosome fragile sites was observed, resulting in changes to gene-expression profiles. Transcription stress near promoters correlated with high GCskew and the accumulation of R-loops at promoter-proximal regions, which localized with chromosomal regions where gains and losses were observed. In the absence of Senataxin, the Cockayne syndrome protein CSB was required for the recruitment of the transcription-coupled repair endonucleases (XPG and XPF) and RAD52 recombination protein to target and resolve transcription bubbles containing R-loops, leading to genomic instability. These results show that transcription stress is an important contributor to SETX mutation-associated chromosome fragility and AOA2.

Real-Time Single-Molecule Studies of RNA Polymerase–Promoter Open Complex Formation Reveal Substantial Heterogeneity Along the Promoter-Opening Pathway

The expression of most bacterial genes commences with the binding of RNA polymerase (RNAP)–σ70 holoenzyme to the promoter DNA. This initial RNAP–promoter closed complex undergoes a series of conformational changes, including the formation of a transcription bubble on the promoter and the loading of template DNA strand into the RNAP active site; these changes lead to the catalytically active open complex (RPO) state. Recent cryo-electron microscopy studies have provided detailed structural insight on the RPO and putative intermediates on its formation pathway. Here, we employ single-molecule fluorescence microscopy to interrogate the conformational dynamics and reaction kinetics during real-time RPO formation on a consensus lac promoter. We find that the promoter opening may proceed rapidly from the closed to open conformation in a single apparent step, or may instead involve a significant intermediate between these states. The formed RPO complexes are also different with respect to their transcription bubble stability. The RNAP cleft loops, and especially the β′ rudder, stabilise the transcription bubble. The RNAP interactions with the promoter upstream sequence (beyond −35) stimulate transcription bubble nucleation and tune the reaction path towards stable forms of the RPO.

Structural origins of Escherichia coli RNA polymerase open promoter complex stability

The first step in gene expression in all organisms requires opening the DNA duplex to expose one strand for templated RNA synthesis. In Escherichia coli, promoter DNA sequence fundamentally determines how fast the RNA polymerase (RNAP) forms "open" complexes (RPo), whether RPo persists for seconds or hours, and how quickly RNAP transitions from initiation to elongation. These rates control promoter strength in vivo, but their structural origins remain largely unknown. Here, we use cryoelectron microscopy to determine the structures of RPo formed de novo at three promoters with widely differing lifetimes at 37 °C: λPR (t1/2 ∼10 h), T7A1 (t1/2 ∼4 min), and a point mutant in λPR (λPR-5C) (t1/2 ∼2 h). Two distinct RPo conformers are populated at λPR, likely representing productive and unproductive forms of RPo observed in solution studies. We find that changes in the sequence and length of DNA in the transcription bubble just upstream of the start site (+1) globally alter the network of DNA-RNAP interactions, base stacking, and strand order in the single-stranded DNA of the transcription bubble; these differences propagate beyond the bubble to upstream and downstream DNA. After expanding the transcription bubble by one base (T7A1), the nontemplate strand "scrunches" inside the active site cleft; the template strand bulges outside the cleft at the upstream edge of the bubble. The structures illustrate how limited sequence changes trigger global alterations in the transcription bubble that modulate the RPo lifetime and affect the subsequent steps of the transcription cycle.

Preparation of E. coli RNA polymerase transcription elongation complexes by selective photoelution from magnetic beads

In vitro studies of transcription frequently require the preparation of defined elongation complexes. Defined transcription elongation complexes (TECs) are typically prepared by constructing an artificial transcription bubble from synthetic oligonucleotides and RNA polymerase. This approach is optimal for diverse applications but is sensitive to nucleic acid length and sequence and is not compatible with systems where promoter-directed initiation or extensive transcription elongation is crucial. To complement scaffold-directed approaches for TEC assembly, I have developed a method for preparing promoter-initiated Escherichia coli TECs using a purification strategy called selective photoelution. This approach combines TEC-dependent sequestration of a biotin–triethylene glycol transcription stall site with photoreversible DNA immobilization to enrich TECs from an in vitro transcription reaction. I show that selective photoelution can be used to purify TECs that contain a 273-bp DNA template and 194-nt structured RNA. Selective photoelution is a straightforward and robust procedure that, in the systems assessed here, generates precisely positioned TECs with >95% purity and >30% yield. TECs prepared by selective photoelution can contain complex nucleic acid sequences and will therefore likely be useful for investigating RNA structure and function in the context of RNA polymerases.

Mathematical Modeling of Transcription Bubble Behavior in the pPF1 Plasmid and its Modified Versions: The Link between the Plasmid Energy Profile and the Direction of Transcription

Mathematical Modeling of Transcription Bubble Behavior in the pPF1 Plasmid and its Modified Versions: The Link between the Plasmid Energy Profile and the Direction of Transcription

Transcription activation by a sliding clamp

Transcription activation of bacteriophage T4 late genes is accomplished by a transcription activation complex containing RNA polymerase (RNAP), the promoter specificity factor gp55, the coactivator gp33, and a universal component of cellular DNA replication, the sliding clamp gp45. Although genetic and biochemical studies have elucidated many aspects of T4 late gene transcription, no precise structure of the transcription machinery in the process is available. Here, we report the cryo-EM structures of a gp55-dependent RNAP-promoter open complex and an intact gp45-dependent transcription activation complex. The structures reveal the interactions between gp55 and the promoter DNA that mediate the recognition of T4 late promoters. In addition to the σR2 homology domain, gp55 has a helix-loop-helix motif that chaperons the template-strand single-stranded DNA of the transcription bubble. Gp33 contacts both RNAP and the upstream double-stranded DNA. Gp45 encircles the DNA and tethers RNAP to it, supporting the idea that gp45 switches the promoter search from three-dimensional diffusion mode to one-dimensional scanning mode.

Double energy profile of pBR322 plasmid

A small circular DNA - plasmid pBR322, is considered as a complex dynamic system where nonlinear conformational perturbations which are often named open states or kinks, can arise and propagate. To describe the internal dynamics of the plasmid we use mathematical model consisting of two coupled sine-Gordon equations that in the average field approximation are transformed to two sine-Gordon independent equations with renormalized coefficients. The first equation describes angular oscillations of nitrous bases of the main chain. The second equation describes angular oscillations of nitrous bases in the complementary chain. As a result, two types of kink-like solutions have been obtained. One type kinks were the solutions of the first equation, and the other kinks were the solutions of the second equation. We calculated the main characteristics of the kink motion, including the time dependences of the kink velocity, coordinates, and total energy. These calculations were performed at the initial velocity equal to 1881 m/s which was chosen to avoid reflections from energy barriers corresponding to CDS-1 and CDS-2. The movement of the kinks was investigated by the method of the double energy profile. The maximum complete set of the DNA dynamic parameters was used to calculate the double profile. To calculate the velocity, energy and trajectory of the kinks, the block method was used. The results obtained made it possible to explain in which region of the plasmid the formation of a transcription bubble is most likely, as well to understand in which direction the bubble will move and the transcription process will go.

Direct binding of TFE\u03b1 opens DNA binding cleft of RNA polymerase

Opening of the DNA binding cleft of cellular RNA polymerase (RNAP) is necessary for transcription initiation but the underlying molecular mechanism is not known. Here, we report on the cryo-electron microscopy structures of the RNAP, RNAP-TFEα binary, and RNAP-TFEα-promoter DNA ternary complexes from archaea, Thermococcus kodakarensis (Tko). The structures reveal that TFEα bridges the RNAP clamp and stalk domains to open the DNA binding cleft. Positioning of promoter DNA into the cleft closes it while maintaining the TFEα interactions with the RNAP mobile modules. The structures and photo-crosslinking results also suggest that the conserved aromatic residue in the extended winged-helix domain of TFEα interacts with promoter DNA to stabilize the transcription bubble. This study provides a structural basis for the functions of TFEα and elucidates the mechanism by which the DNA binding cleft is opened during transcription initiation in the stalk-containing RNAPs, including archaeal and eukaryotic RNAPs.

CueR activates transcription through a DNA distortion mechanism.

The MerR-family transcription factors (TFs) are a large group of bacterial proteins responding to cellular metal ions and multiple antibiotics by binding within central RNA polymerase-binding regions of a promoter. While most TFs alter transcription through protein-protein interactions, MerR TFs are capable of reshaping promoter DNA. To address the question of which mechanism prevails, we determined two cryo-EM structures of transcription activation complexes (TAC) comprising Escherichia coli CueR (a prototype MerR TF), RNAP holoenzyme and promoter DNA. The structures reveal that this TF promotes productive promoter-polymerase association without canonical protein-protein contacts seen between other activator proteins and RNAP. Instead, CueR realigns the key promoter elements in the transcription activation complex by clamp-like protein-DNA interactions: these induce four distinct kinks that ultimately position the -10 element for formation of the transcription bubble. These structural and biochemical results provide strong support for the DNA distortion paradigm of allosteric transcriptional control by MerR TFs.