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Graphical abstract, Highlights • The formation of single RNA polymerase–promoter open complexes probed in real-time. • Substantial heterogeneity was found along the promoter-opening pathway. • Branched steps include template strand loading into the active site. • Branched steps also include transcription bubble stabilisation. • RNA polymerase rudder loop stabilises open transcription bubble., 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.

In this study we used the kink solutions of the nonlinear sine-Gordon equation to apply methods of mathematical modeling to investigate the dynamics of the transcription bubble in the pPF1 plasmid. Based on the calculated energy profile for the pPF1 plasmid and its modified versions, it was shown that the minimum potential energy of the kink formation or transcription bubble nucleation corresponds to the region between the genes of the Egfp and mCherry proteins. The insertion of homogeneous sequences into the region between Egfp and mCherry showed that the kink is more likely to be activated in polyT or polyC compared to polyA or polyG, which indicates the dependence of nucleation of the transcription bubble on the molecular weight of base pairs. For insertions into the region between Egfp and mCherry of small fragments of the native sequence of Escherichia coli, the model identifies the DNA strands with the highest probability of nucleation of the transcription bubble and, accordingly, determines the direction (towards the Egfp or mCherry gene) of transcription, indicating a link between the direction of transcription and the energy profile of the 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.

Transcription is punctuated by RNA polymerase (RNAP) pausing. These pauses provide time for diverse regulatory events that can modulate gene expression. Transcription elongation factors dramatically affect RNAP pausing in vitro, but the genome-wide role of such factors on pausing has not been examined. Using native elongating transcript sequencing followed by RNase digestion (RNET-seq), we analyzed RNAP pausing in Bacillus subtilis genome-wide and identified an extensive role of NusG in pausing. This universally conserved transcription elongation factor is known as Spt5 in archaeal and eukaryotic organisms. B. subtilis NusG shifts RNAP to the posttranslocation register and induces pausing at 1,600 sites containing a consensus TTNTTT motif in the nontemplate DNA strand within the paused transcription bubble. The TTNTTT motif is necessary but not sufficient for NusG-dependent pausing. Approximately one-fourth of these pause sites were localized to untranslated regions and could participate in posttranscription initiation control of gene expression as was previously shown for tlrB and the trpEDCFBA operon. Most of the remaining pause sites were identified in protein-coding sequences. NusG-dependent pausing was confirmed for all 10 pause sites that we tested in vitro. Putative pause hairpins were identified for 225 of the 342 strongest NusG-dependent pause sites, and some of these hairpins were shown to function in vitro. NusG-dependent pausing in the ribD riboswitch provides time for cotranscriptional binding of flavin mononucleotide, which decreases the concentration required for termination upstream of the ribD coding sequence. Our phylogenetic analysis implicates NusG-dependent pausing as a widespread mechanism in bacteria.

The structurally homologous Mtf1 and TFB2M proteins serve as transcription initiation factors of mitochondrial RNA polymerases in Saccharomyces cerevisiae and humans, respectively. These transcription factors directly interact with the nontemplate strand of the transcription bubble to drive promoter melting. Given the key roles of Mtf1 and TFB2M in promoter-specific transcription initiation, it can be expected that the DNA binding activity of the mitochondrial transcription factors is regulated to prevent DNA binding at inappropriate times. However, little information is available on how mitochondrial DNA transcription is regulated. While studying C-terminal (C-tail) deletion mutants of Mtf1 and TFB2M, we stumbled upon a finding that suggested that the flexible C-tail region of these factors autoregulates their DNA binding activity. Quantitative DNA binding studies with fluorescence anisotropy-based titrations revealed that Mtf1 with an intact C-tail has no affinity for DNA but deletion of the C-tail greatly increases Mtf1's DNA binding affinity. Similar observations were made with TFB2M, although autoinhibition by the C-tail of TFB2M was not as complete as in Mtf1. Analysis of available TFB2M structures disclosed that the C-tail engages in intramolecular interactions with the DNA binding groove in the free factor, which, we propose, inhibits its DNA binding activity. Further experiments showed that RNA polymerase relieves this autoinhibition by interacting with the C-tail and engaging it in complex formation. In conclusion, our biochemical and structural analyses reveal autoinhibitory and activation mechanisms of mitochondrial transcription factors that regulate their DNA binding activities and aid in specific assembly of transcription initiation complexes.

Single molecule FRET (smFRET) is a useful tool for studying biomolecular sub-populations and their dynamics. Advanced smFRET-based techniques often track multiple parameters simultaneously, increasing the information content of the measurement. Photon-by-photon hidden Markov modelling (H2MM) is a smFRET analysis tool that quantifies FRET dynamics of single biomolecules, even if they occur in sub-milliseconds. However, sub-populations can be characterized by additional experimentally-derived parameters other than the FRET efficiency. We introduce multi-parameter H2MM (mpH2MM) that identifies sub-populations and their transition dynamics based on multiple experimentally-derived parameters, simultaneously. We show the use of this tool in deciphering the number of underlying sub-populations, their mean characteristics and the rate constants of their transitions for a DNA hairpin exhibiting milliseconds FRET dynamics, and for the RNA polymerase promoter open complex exhibiting sub-millisecond FRET dynamics of the transcription bubble. Overall, we show that using mpH2MM facilitates the identification and quantification of biomolecular sub-populations in smFRET measurements that are otherwise difficult to identify. Finally we provide the means to use mpH2MM in analyzing FRET dynamics in advanced multi-color smFRET-based measurements.

The first step of 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 cryo-electron microscopy to determine structures of RPo formed de novo at three promoters with widely differing lifetimes at 37°C: λPR (t1/2 ∼ 10 hours), T7A1 (t1/2 ∼ 4 minutes), and a point mutant in λPR (λPR-5C) (t1/2 ∼ 2 hours). 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 RPo lifetime and affect the subsequent steps of the transcription cycle.

Papers concerning transcription, and its regulation at promoters, abound in the scientific literature, but reports about initiation, defined as the moment that the first 3′−5′phosphodiester bond of a new transcript is forged, are scarce. In PNAS, Skalenko et al. (1), working with the bacterial multisubunit DNA-dependent RNA polymerase (RNAP), combine high-throughput DNA base-sequencing methods with structural analysis, to provide insights into how RNAP manages the two partner molecules needed to create that first bond, as each new RNA transcript is born. Textbooks paint a deceptively simple picture of transcript initiation: At the majority of bacterial promoters, the RNAP σ (sigma) subunit orchestrates promoter recognition, and then drives the opening of just over one turn of the duplex DNA to form a “transcription bubble,” which is essential for the template strand to be read (2). This is done almost single-handedly by domain 2 of the σ subunit, which makes specific base-dependent interactions with the nontemplate strand of the promoter DNA, and this allows the single-stranded template strand access to the RNAP catalytic site (3⇓–5). The textbook tells us that, then, two nucleoside triphosphates (NTPs) are selected by base pairing with the template strand bases, denoted “TSS” (for transcription start site) and “TSS+1” (one base downstream), and, once in the product and addition sites, the RNAP catalytic activity facilitates phosphodiester bond formation. However, as so often, the reality is not quite so simple, and, over the past decade, Bryce Nickels, Richard Ebright, and their colleagues at Rutgers University have produced a stellar series of papers that, step by step, have unveiled the mysteries of initiation. The first complication arises from inequalities in how RNAP manages the two DNA strands in the transcription bubble (Fig. 1). Prior to initiation, the so-called −10 hexamer element (consensus 5′-TATAAT-3′) in the nontemplate strand … [↵][1]1Email: s.j.w.busby{at}bham.ac.uk. [1]: #xref-corresp-1-1

Endogenous RNA transcription characterizes double-stranded RNA (dsRNA) viruses in the Reoviridae, a family that is exemplified by its simple, single-shelled member cytoplasmic polyhedrosis virus (CPV). Because of the lack of in situ structures of the intermediate stages of RNA-dependent RNA polymerase (RdRp) during transcription, it is poorly understood how RdRp detects environmental cues and internal transcriptional states to initiate and coordinate repeated cycles of transcript production inside the capsid. Here, we captured five high-resolution (2.8–3.5 A) RdRp–RNA in situ structures—representing quiescent, initiation, early elongation, elongation and abortive states—under seven experimental conditions of CPV. We observed the ‘Y’-form initial RNA fork in the initiation state and the complete transcription bubble in the elongation state. These structures reveal that de novo RNA transcription involves three major conformational changes during state transitions. Our results support an ouroboros model for endogenous conservative transcription in dsRNA viruses. In situ cryo-electron microscopy structures of five intermediate states of the RNA-dependent RNA polymerase of cytoplasmic polyhedrosis virus reveals how repeated cycles of double-stranded RNA genome transcription are regulated within viral particles.

Activation-induced deoxycytidine deaminase (AID) initiates somatic hypermutation (SHM) in immunoglobulin variable (IgV) genes to produce high-affinity antibodies. SHM requires IgV transcription by RNA polymerase II (Pol II). A eukaryotic transcription system including AID has not been reported previously. Here, we reconstitute AID-catalyzed deamination during Pol II transcription elongation in conjunction with DSIF transcription factor. C→T mutations occur at similar frequencies on non-transcribed strand (NTS) and transcribed strand (TS) DNA. In contrast, bacteriophage T7 Pol generates NTS mutations predominantly. AID-Pol II mutations are strongly favored in WRC and WGCW overlapping hot motifs (W = A or T, R = A or G) on both DNA strands. Single mutations occur on 70% of transcribed DNA clones. Mutations are correlated over a 15 nt distance in multiply mutated clones, suggesting that deaminations are catalyzed processively within a stalled or backtracked transcription bubble. Site-by-site comparisons for biochemical and human memory B-cell mutational spectra in an IGHV3-23*01 target show strongly favored deaminations occurring in the antigen-binding complementarity determining regions (CDR) compared to the framework regions (FW). By exhibiting consistency with B-cell SHM, our in vitro data suggest that biochemically defined reconstituted Pol II transcription systems can be used to investigate how, when and where AID is targeted.

Transcribing and replicating a double-stranded genome require protein modules to unwind, transcribe/replicate nucleic acid substrates, and release products. Here we present in situ cryo-electron microscopy structures of rotavirus dsRNA-dependent RNA polymerase (RdRp) in two states pertaining to transcription. In addition to the previously discovered universal “hand-shaped” polymerase core domain shared by DNA polymerases and telomerases, our results show the function of N- and C-terminal domains of RdRp: the former opens the genome duplex to isolate the template strand; the latter splits the emerging template-transcript hybrid, guides genome reannealing to form a transcription bubble, and opens a capsid shell protein (CSP) to release the transcript. These two “helicase” domains also extensively interact with CSP, which has a switchable N-terminal helix that, like cellular transcriptional factors, either inhibits or promotes RdRp activity. The in situ structures of RdRp, CSP, and RNA in action inform mechanisms of not only transcription, but also replication., Rotaviruses are of great medical significance because they cause gastroenteritis in children. Here the authors provide insights into the mechanism of viral mRNA transcription by determining the in situ cryo-EM structures of a working rotavirus’ RNA-dependent-RNA polymerase, which is of interest for antiviral drug design.

Extrinsic transcription termination typically involves remodeling of RNA polymerase by an accessory helicase. In yeast this is accomplished by the Sen1 helicase homologous to human senataxin (SETX). To gain insight into these processes we develop a DNA scaffold construct compatible with magnetic-trapping assays and from which S. cerevisiae RNA polymerase II (Pol II), as well as E. coli RNA polymerase (ecRNAP), can efficiently initiate transcription without transcription factors, elongate, and undergo extrinsic termination. By stalling Pol II TECs on the construct we can monitor Sen1-induced termination in real-time, revealing the formation of an intermediate in which the Pol II transcription bubble appears half-rewound. This intermediate requires ~40 sec to form and lasts ~20 sec prior to final dissociation of the stalled Pol II. The experiments enabled by the scaffold construct permit detailed statistical and kinetic analysis of Pol II interactions with a range of cofactors in a multi-round, high-throughput fashion., Yeast’s Sen1 helicase is involved in the suppression of antisense transcription from bidirectional eukaryotic promoters. Here authors develop and utilize a quantitative single-molecule assay reporting on the kinetics of extrinsic eukaryotic transcription termination by the Sen1 helicase and a reaction intermediate in which the Pol II transcription bubble appears half-rewound.

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. We find that the RPO pathway is branched, generating RPO complexes with different stabilities. 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. The mechanistic heterogeneity of the RPO pathway may be a prerequisite for its regulation since such heterogeneity allows the amplification of small promoter sequence or transcription-factor-dependent changes in the free energy profile of the RPO pathway to large differences in transcription efficiency.

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.

Transcription initiation requires assembly of the RNA polymerase II (Pol II) pre-initiation complex (PIC) and opening of promoter DNA. Here, we present the long-sought high-resolution structure of the yeast PIC and define the mechanism of initial DNA opening. We trap the PIC in an intermediate state that contains half a turn of open DNA located 30-35 base pairs downstream of the TATA box. The initially opened DNA region is flanked and stabilized by the polymerase "clamp head loop" and the TFIIF "charged region" that both contribute to promoter-initiated transcription. TFIIE facilitates initiation by buttressing the clamp head loop and by regulating the TFIIH translocase. The initial DNA bubble is then extended in the upstream direction, leading to the open promoter complex and enabling start-site scanning and RNA synthesis. This unique mechanism of DNA opening may permit more intricate regulation than in the Pol I and Pol III systems.

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., Transcription activation of late genes in T4 bacteriophage requires the promoter specificity factor gp55, the coactivator gp33 and the sliding clamp gp45. Here, the authors provide structural insights into gp45- dependent transcription activation by determining the cryo-EM structures of a gp55-dependent RNA polymerase (RNAP)-promoter open complex and of an intact gp45-dependent transcription activation complex.

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., How clamp conformation is regulated in the transcription cycle of stalk-containing archaeal and eukaryotic RNA polymerase (RNAP) systems is still not well understood. Here, the authors combine cryo-EM, X-ray crystallography and photo-crosslinking assays to structurally characterise RNAP, the RNAP-TFEα binary and RNAP-TFEα-promoter DNA ternary complexes from the archaea Thermococcus kodakarensis and enables them to describe the dynamic conformational changes of the general transcription factor TFEα and RNAP during the early stage of transcription cycle.

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. Structural analysis of transcription activation complex comprising E. coli transcription factor CueR, RNAP holoenzyme and promoter DNA reveals that CueR distorts the DNA conformation to promote the association of promoter with polymerase.

Transcription initiation requires formation of the open promoter complex, RPo. To generate RPo, RNA polymerase (RNAP) unwinds the DNA duplex to form the transcription bubble and loads the DNA into the RNAP active site. RPo formation is a multi-step process with transient intermediates of unknown structure. We used single particle cryo-electron microscopy to visualize seven intermediates containing Escherichia coli RNAP with the transcription factor TraR en route to forming RPo. The structures span the RPo formation pathway from initial recognition of the duplex promoter in a closed complex to the final RPo. The structures and supporting biochemical data define RNAP and promoter DNA conformational changes that delineate steps on the pathway, including previously undetected transient promoter-RNAP interactions that contribute to populating the intermediates but do not occur in RPo. Our work provides a structural basis for understanding RPo formation and its regulation, a major checkpoint in gene expression throughout evolution.

The structurally homologous Mtf1 and TFB2M proteins serve as transcription initiation factors of theSaccharomyces cerevisiaeand human mitochondrial RNA polymerases, respectively. These transcription factors directly interact with the non-template strand of the transcription bubble to drive promoter melting. Given the key roles of Mtf1 and TFB2M in promoter-specific transcription initiation, it is expected that the DNA binding activity of the mitochondrial transcription factors would be regulated to prevent DNA binding at inappropriate times. However, there is little information on how mitochondrial DNA transcription is regulated. While studying the C-tail deletion mutants of Mtf1 and TFB2M, we stumbled upon a new finding that suggested that the flexible C-tail region of these factors autoregulates their DNA binding activity. Quantitative DNA binding studies with fluorescence anisotropy-based titrations show that Mtf1 with an intact C-tail has no affinity for the DNA but the deletion of C-tail greatly increases the DNA binding affinity. Similar observations were made with TFB2M, although autoinhibition by the C-tail of TFB2M was not as absolute as in Mtf1. Analysis of available TFB2M structures show that the C-tail makes intramolecular interactions with the DNA binding groove in the free factor, which we propose masks the DNA binding activity. Further studies show that the RNA polymerase relieves autoinhibition by interacting with the C-tail and engaging it in complex formation. Thus, our biochemical and structural analysis identify previously unknown autoinhibitory and activation mechanisms of mitochondrial transcription factors that regulate the DNA binding activity and aid in specific assembly of the initiation complexes.

In this work, we study numerically the influence of the DNA torque on the movement of transcription bubbles in the potential field formed by the sequence of plasmid PTTQ18. To imitate the movement, we apply a modified sine-Gordon equation with the two additional terms that describe the effects of dissipation and the action of the DNA torsion torque, and with the coefficients that depend on the sequence of bases. We obtain the trajectories of the transcription bubbles and investigate the dependence of the trajectories on the initial bubble velocity and the DNA torsion torque. It is shown that not the initial bubble velocity but the DNA torsion torque governs the trajectories of the transcription bubbles.

Universally conserved NusG/Spt5 factors reduce RNA polymerase pausing and arrest. In a widely accepted model, these proteins bridge the RNA polymerase clamp and lobe domains across the DNA channel, inhibiting the clamp opening to promote pause-free RNA synthesis. However, recent structures of paused transcription elongation complexes show that the clamp does not open and suggest alternative mechanisms of antipausing. Among these mechanisms, direct contacts of NusG/Spt5 proteins with the nontemplate DNA in the transcription bubble have been proposed to prevent unproductive DNA conformations and thus inhibit arrest. We used Escherichia coli RfaH, whose interactions with DNA are best characterized, to test this idea. We report that RfaH stabilizes the upstream edge of the transcription bubble, favoring forward translocation, and protects the upstream duplex DNA from exonuclease cleavage. Modeling suggests that RfaH loops the nontemplate DNA around its surface and restricts the upstream DNA duplex mobility. Strikingly, we show that RfaH-induced DNA protection and antipausing activity can be mimicked by shortening the nontemplate strand in elongation complexes assembled on synthetic scaffolds. We propose that remodeling of the nontemplate DNA controls recruitment of regulatory factors and R-loop formation during transcription elongation across all life.

A minimal set of general transcription factors (GTFs) is required for RNA polymerase II (pol II) to initiate transcription at promoters. For all eukaryotes, from yeast to mammals, the GTFs include TFIIA, TFIIB, TFIIE, TFIIF, TFIIH, and the TATA box binding protein TBP (1⇓–3). When assembled at the promoter with pol II, the resulting preinitiation complex (PIC) spans 60 to 65 bp along the template DNA (2, 4). For RNA polymerase to advance into full transcript elongation it must escape from the PIC, which is anchored by many specific protein–DNA interactions. The mechanism through which the PIC/initiating complex transitions to the committed elongation complex represents a major unanswered challenge in transcription biochemistry. In PNAS, Fujiwara et al. (5) report the application of a significantly improved yeast pol II in vitro transcription system to provide important insight into pol II promoter clearance. Our current understanding of promoter clearance is based primarily on work in mammalian systems. A crucial feature is the size and location of the unpaired segment of template DNA (the transcription bubble) as transcription initiates and proceeds. In mammals, the template strands are initially unwound by the ATP-dependent XPB helicase of TFIIH, with the bubble extending about 9 nt upstream of the transcription start site (TSS) (6). As transcription initiates and pol II extends the nascent RNA, the upstream end of the bubble remains in place while the downstream edge advances with transcription until the bubble reaches ∼18 nt, driven in part by the action of XPB. When transcript elongation continues beyond this point, the upstream segment of the bubble abruptly closes, reducing the bubble to the ∼10-bp segment characteristic of elongation complexes (6, 7). This partial bubble collapse coincides with the stabilization of the transcription complex (7) and loss of the requirement for … [↵][1]1Email: lused{at}ccf.org. [1]: #xref-corresp-1-1

RNA polymerase (RNAP) is a major target of gene regulation. Thermus thermophilus bacteriophage P23–45 encodes two RNAP binding proteins, gp39 and gp76, which shut off host gene transcription while allowing orderly transcription of phage genes. We previously reported the structure of the T. thermophilus RNAP•σA holoenzyme complexed with gp39. Here, we solved the structure of the RNAP•σA holoenzyme bound with both gp39 and gp76, which revealed an unprecedented inhibition mechanism by gp76. The acidic protein gp76 binds within the RNAP cleft and occupies the path of the template DNA strand at positions –11 to –4, relative to the transcription start site at +1. Thus, gp76 obstructs the formation of an open promoter complex and prevents transcription by T. thermophilus RNAP from most host promoters. gp76 is less inhibitory for phage transcription, as tighter RNAP interaction with the phage promoters allows the template DNA to compete with gp76 for the common binding site. gp76 also inhibits Escherichia coli RNAP highlighting the template–DNA binding site as a new target site for developing antibacterial agents.

Transcription has classically been considered a potential threat to genome integrity. Collision between transcription and DNA replication machinery, and retention of DNA:RNA hybrids, may result in genome instability. On the other hand, it has been proposed that active genes repair faster and preferentially via homologous recombination. Moreover, while canonical transcription is inhibited in the proximity of DNA double-strand breaks, a growing body of evidence supports active non-canonical transcription at DNA damage sites. Small non-coding RNAs accumulate at DNA double-strand break sites in mammals and other organisms, and are involved in DNA damage signaling and repair. Furthermore, RNA binding proteins are recruited to DNA damage sites and participate in the DNA damage response. Here, we discuss the impact of transcription on genome stability, the role of RNA binding proteins at DNA damage sites, and the function of small non-coding RNAs generated upon damage in the signaling and repair of DNA lesions.

Transcription machinery remains steadfast Eukaryotic transcription of mRNA is a multistep process mediated by RNA polymerase II (Pol II). Pol II combines with several other factors to form an elongation complex that promotes transcription elongation. Ehara et al. determined the high-resolution structure of the elongation complex by means of x-ray crystallography and cryo-electron microscopy (see the Perspective by Fouqueau and Werner). Multiple elongation factors are distributed on a wide surface of Pol II and establish an RNA exit path and DNA entry or exit tunnels, which facilitate nascent transcript transfer and DNA unwinding or rewinding. The Pol II elongation complex thus adopts a stable architecture suitable for processive transcription. Science , this issue p. 921 ; see also p. 871

RNA polymerase (RNAP) is the central motor of gene expression since it governs the process of transcription. In prokaryotes, this holoenzyme is formed by the RNAP core and a sigma factor. After approaching and binding the specific promoter site on the DNA, the holoenzyme-promoter complex undergoes several conformational transitions that allow unwinding and opening of the DNA duplex. Once the first DNA basepairs (∼10 bp) are transcribed in an initial transcription process, the enzyme unbinds from the promoter and proceeds downstream along the DNA while continuously opening the helix and polymerizing the ribonucleotides in correspondence with the template DNA sequence. When the gene is transcribed into RNA, the process generally is terminated and RNAP unbinds from the DNA. The first step of transcription-initiation, is considered the rate-limiting step of the entire process. This review focuses on the single-molecule studies that try to reveal the key steps in the initiation phase of bacterial transcription. Such single-molecule studies have, for example, allowed real-time observations of the RNAP target search mechanism, a mechanism still under debate. Moreover, single-molecule studies using Forster Resonance Energy Transfer (FRET) revealed the conformational changes that the enzyme undergoes during initiation. Force-based techniques such as scanning force microscopy and magnetic tweezers allowed quantification of the energy that drives the RNAP translocation along DNA and its dynamics. In addition to these in vitro experiments, single particle tracking in vivo has provided a direct quantification of the relative populations in each phase of transcription and their locations within the cell.

G-quadruplex (GQ) structures are implicated in important physiological and pathological processes. Millions of GQ-forming motifs are enriched near transcription start sites (TSSs) of animal genes. Transcription can induce the formation of GQs, which in turn regulate transcription. The kinetics of the formation and persistence of GQs in transcription is crucial for the role they play but has not yet been explored. We established a method based on the fluorescence resonance energy transfer (FRET) technique to monitor in real-time the cotranscriptional formation and post-transcriptional persistence of GQs in DNA. Using a T7 transcription model, we demonstrate that a representative intramolecular DNA GQ and DNA:RNA hybrid GQ promptly form in proportion to transcription activity and, once formed, are maintained for hours or longer at physiological temperature even after transcription is stopped. Both their formation and persistence strongly depend on R-loop, a DNA:RNA hybrid duplex formed during transcription. Enzymatic removal of R-loop dramatically slows their formation and accelerates their unfolding. These results suggest that a transcription event is promptly read-out by GQ-forming motifs and the GQ formed can either perform regulation in fast response to transcription and/or memorized in DNA to mediate time-delayed regulation under the control of RNA metabolism and GQ-resolving activity. Alternatively, GQs need to be timely resolved to warrant success of translocating activities such as replication. The kinetic characteristics of GQs and its connection with the R-loop may have implications in transcription regulation, signal transduction, G-quadruplex processing, and genome stability.

Non-canonical DNA structures can obstruct transcription. This transcription blockage could have various biological consequences, including genomic instability and gratuitous transcription-coupled repair. Among potential structures causing transcription blockage are Holliday junctions (HJs), which can be generated as intermediates in homologous recombination or during processing of stalled replication forks. Of particular interest is the double Holliday junction (DHJ), which contains two HJs. Topological considerations impose the constraint that the total number of helical turns in the DNA duplexes between the junctions cannot be altered as long as the flanking DNA duplexes are intact. Thus, the DHJ structure should strongly resist transient unwinding during transcription; consequently, it is predicted to cause significantly stronger blockage than single HJ structures. The patterns of transcription blockage obtained for RNA polymerase II transcription in HeLa cell nuclear extracts were in accordance with this prediction. However, we did not detect transcription blockage with purified T7 phage RNA polymerase; we discuss a possible explanation for this difference. In general, our findings implicate naturally occurring Holliday junctions in transcription arrest.

A method for the physical attachment of folded RNA libraries to their encoding DNA is presented as a way to circumvent the reverse transcription step during systematic evolution of RNA ligands by exponential enrichment (RNA-SELEX). A DNA library is modified with one isodC base to stall T7 polymerase and a 5′ “capture strand” which anneals to the nascent RNA transcript. This method is validated in a selection of RNA aptamers against human α-thrombin with dissociation constants in the low nanomolar range. This method will be useful in the discovery of RNA aptamers and ribozymes containing base modifications that make them resistant to accurate reverse transcription.

At a transcriptional pause site, RNA polymerase (RNAP) takes significantly longer than average to transcribe the nucleotide before moving on to the next position. At the single-molecule level this process is stochastic, while at the ensemble level it plays a variety of important roles in biological systems. The pause signal is complex and invokes interplay between a range of mechanisms. Among these factors are: non-canonical transcription events – such as backtracking and hypertranslocation; the catalytically inactive intermediate state hypothesised to act as a precursor to backtracking; the energetic configuration of basepairing within the DNA/RNA hybrid and of those flanking the transcription bubble; and the structure of the nascent mRNA. There are a variety of plausible models and hypotheses but it is unclear which explanations are better.We performed a systematic comparison of 128 kinetic models of transcription using approximate Bayesian computation. Under this Bayesian framework, models and their parameters were assessed by their ability to predict the locations of pause sites in theE.coligenome.These results suggest that the structural parameters governing the transcription bubble, and the dynamics of the transcription bubble during translocation, play significant roles in pausing. This is consistent with a model where the relative Gibbs energies between the pre and posttranslocated positions, and the rate of translocation between the two, is the primary factor behind invoking transcriptional pausing. Whereas, hypertranslocation, backtracking, and the intermediate state are not required to predict the locations of transcriptional pause sites. Finally, we compared the predictive power of these kinetic models to that of a non-explanatory statistical model. The latter approach has significantly greater predictive power (AUC = 0.89 cf. 0.73), suggesting that, while current models of transcription contain a moderate degree of predictive power, a much greater quantitative understanding of transcriptional pausing is required to rival that of a sequence motif.Author summaryTranscription involves the copying of a DNA template into messenger RNA (mRNA). This reaction is implemented by RNA polymerase (RNAP) successively incorporating nucleotides onto the mRNA. At a transcriptional pause site, RNAP takes significantly longer than average to incorporate the nucleotide. A model which can not only predict the locations of pause sites in a DNA template, but also explainhoworwhythey are pause sites, is sought after.Transcriptional pausing emerges from cooperation between several mechanisms. These mechanisms include non-canonical RNAP reactions; and the thermodynamic properties of DNA and mRNA. There are many hypotheses and kinetic models of transcription but it is unclear which hypotheses and models are required to predict and explain transcriptional pausing.We have developed a rigorous statistical framework for inferring model parameters and comparing hypotheses. By applying this framework to published pause-site data, we compared 128 kinetic models of transcription with the aim of finding the best models for predicting the locations of pause sites. This analysis offered insights into mechanisms of transcriptional pausing. However, the predictive power of these models lacks compared with non-explanatory statistical models - suggesting the data contains more information than can be satisfied by current quantitative understandings of transcriptional pausing.

Type III-A CRISPR-Cas systems are prokaryotic RNA-guided adaptive immune systems that use a protein-RNA complex, Csm, for transcription-dependent immunity against foreign DNA. Csm can cleave RNA and single-stranded DNA (ssDNA), but whether it targets one or both nucleic acids during transcription elongation is unknown. Here, we show that binding of a Thermus thermophilus (T. thermophilus) Csm (TthCsm) to a nascent transcript in a transcription elongation complex (TEC) promotes tethering but not direct contact of TthCsm with RNA polymerase (RNAP). Biochemical experiments show that both TthCsm and Staphylococcus epidermidis (S. epidermidis) Csm (SepCsm) cleave RNA transcripts, but not ssDNA, at the transcription bubble. Taken together, these results suggest that Type III systems primarily target transcripts, instead of unwound ssDNA in TECs, for immunity against double-stranded DNA (dsDNA) phages and plasmids. This reveals similarities between Csm and eukaryotic RNA interference, which also uses RNA-guided RNA targeting to silence actively transcribed genes., Type III CRISPR-Cas systems are able to target transcriptionally active DNA sequences in phages and plasmids. Here, the authors reveal the mechanism of the target nucleic acid preference of Type III-A CRISPR-Cas complexes at the transcription bubble by a combination of structural and biochemical approaches.

Cellular RNA polymerase is a multi-subunit macromolecular assembly responsible for gene transcription, a highly regulated process conserved from bacteria to humans. In bacteria, sigma factors are employed to mediate gene-specific expression in response to a variety of environmental conditions. The major variant σ factor, σ54, has a specific role in stress responses. Unlike σ70-dependent transcription, which often can spontaneously proceed to initiation, σ54-dependent transcription requires an additional ATPase protein for activation. As a result, structures of a number of distinct functional states during the dynamic process of transcription initiation have been captured using the σ54 system with both x-ray crystallography and cryo electron microscopy, furthering our understanding of σ54-dependent transcription initiation and DNA opening. Comparisons with σ70 and eukaryotic polymerases reveal unique and common features during transcription initiation., Graphical Abstract Unlabelled Image, Highlights • Mechanism of transcription inhibition by σ54 • Coupled load and unwind model for transcription initiation • Role of bacterial enhancer-binding proteins in isomerization to the open complex • Comparison of structural and functional differences between σ70 and σ54