Initiation-elongation Transition Research Articles

Three-step mechanism of promoter escape by RNA polymerase II

The transition from transcription initiation to elongation is highly regulated in human cells but remains incompletely understood at the structural level. In particular, it is unclear how interactions between RNA polymerase II (RNA Pol II) and initiation factors are broken to enable promoter escape. Here, we reconstitute RNA Pol II promoter escape invitro and determine high-resolution structures of initially transcribing complexes containing 8-, 10-, and 12-nt ordered RNAs and two elongation complexes containing 14-nt RNAs. We suggest that promoter escape occurs in three major steps. First, the growing RNA displaces the B-reader element of the initiation factor TFIIB without evicting TFIIB. Second, the rewinding of the transcription bubble coincides with the eviction of TFIIA, TFIIB, and TBP. Third, the binding of DSIF and NELF facilitates TFIIE and TFIIH dissociation, establishing the paused elongation complex. This three-step model for promoter escape fills a gap in our understanding of the initiation-elongation transition of RNA Pol II transcription.

Structural visualization of de novo transcription initiation by Saccharomyces cerevisiae RNA polymerase II

Previous structural studies of the initiation-elongation transition of RNA polymerase II (pol II) transcription have relied on the use of synthetic oligonucleotides, often artificially discontinuous to capture pol II in the initiating state. Here, we report multiple structures of initiation complexes converted de novo from a 33-subunit yeast pre-initiation complex (PIC) through catalytic activities and subsequently stalled at different template positions. We determine that PICs in the initially transcribing complex (ITC) can synthesize a transcript of ∼26 nucleotides before transitioning to an elongation complex (EC) as determined by the loss of general transcription factors (GTFs). Unexpectedly, transition to an EC was greatly accelerated when an ITC encountered a downstream EC stalled at promoter proximal regions and resulted in a collided head-to-end dimeric EC complex. Our structural analysis reveals a dynamic state of TFIIH, the largest of GTFs, in PIC/ITC with distinct functional consequences at multiple steps on the pathway to elongation.

Single-Molecule Study Reveals the Conformational Dynamics During Initiation-Elongation Transition in Mitochondrial Transcription

Single-Molecule Study Reveals the Conformational Dynamics During Initiation-Elongation Transition in Mitochondrial Transcription

Sub1/PC4, a multifaceted factor: from transcription to genome stability.

Yeast Sub1 and human PC4, two DNA-binding proteins, were originally identified as transcriptional coactivators with a role during transcription preinitiation/initiation. Indeed, Sub1 is a PIC component, and both PC4 and Sub1 also influence the initiation-elongation transition. Moreover, in the specific case of Sub1, it has been clearly reported that it influences processes downstream during mRNA biogenesis, such as transcription elongation, splicing and termination, and even RNAPII phosphorylation/dephosphorylation. Although Sub1 mechanism of action has been mostly unknown up to date, thanks to the recent finding that Sub1 directly interacts with the RNAPII stalk domain, we can envision how it can modulate so many processes. In addition, Sub1 and PC4 participate in RNAPIII transcription as well, and much additional evidence indicates an evolutionarily conserved role for Sub1 and PC4 in the maintenance of genome stability. In this regard, the most novel function of Sub1 and PC4 has been related to the ability of these proteins to bind G-quadruplex DNA structures that may arise as a consequence of the transcription process.

Single-Molecule Real-Time 3D Imaging of the Transcription Cycle by Modulation Interferometry

Many essential cellular processes, such as gene control, employ elaborate mechanisms involving the coordination of large, multi-component molecular assemblies. Few structural biology tools presently have the combined spatial-temporal resolution and molecular specificity required to capture the movement, conformational changes, and subunit association-dissociation kinetics, three fundamental elements of how such intricate molecular machines work. Here, we report a 3D single-molecule super-resolution imaging study using modulation interferometry and phase-sensitive detection that achieves <2nm axial localization precision, well below thefew-nanometer-sized individual protein components. To illustrate the capability of this technique in probing the dynamics of complex macromolecular machines, we visualize the movement of individual multi-subunit E.coli RNA polymerases through the complete transcription cycle, dissect the kinetics of the initiation-elongation transition, and determine the fate of σ70 initiation factors during promoter escape. Modulation interferometry sets the stage for single-molecule studies of several hitherto difficult-to-investigate multi-molecular transactions that underlie genome regulation.

E. Coli RNA Polymerase Pauses during Initial Transcription

We present recent work that applies smFRET techniques to the early stages of bacterial transcription in which DNA is transcribed into RNA, resulting in a FRET change as a DNA template is transcribed. The mechanism of initial transcription is unclear, mainly due to challenges arising from transient intermediates and molecular heterogeneity. Our work has revealed that RNA polymerase pauses after producing a 6-mer RNA, and that the pause can serve as a regulatory checkpoint.Both DNA template sequence and polymerase structure can modulate this pause. Recent work on elongating RNA polymerase by multiple groups identified a sequence element (G-10Y-1G+1) that promotes pausing during elongation. We have found that the ‘YG’ portion of this sequence can induce a long-lived pause (∼20s) during early initiation on a lac promoter. We also identify a structural region of the polymerase, σ3.2, which contains a loop blocking the RNA-exit channel, as a pausing determinant. This region blocks RNA extension but also forms stabilizing contacts with the nascent RNA that may serve to limit transcript dissociation.The observation of a ‘YG’-induced pause during both initiation and elongation suggests that its structural basis is conserved in the initiation-elongation transition -- in agreement with the idea that the ‘YG’ motif biases the polymerase towards the pre-translocated state. Indeed, our results with a σ3.2 deletion mutant suggest that its inability to maintain the translocational register of the nascent RNA transcript prevents ‘YG’-induced pausing.We have used the information obtained on pausing to develop a new working model for initial transcription. In addition to the insight we have gained into the dynamics and structure of transient intermediates, this project is an excellent case study in how single-molecule techniques can inform the design of classical biochemical experiments – and vice versa.

The Ssu72 Phosphatase Mediates the RNA Polymerase II Initiation-Elongation Transition

Transitions between the different stages of the RNAPII transcription cycle involve the recruitment and exchange of factors, including mRNA capping enzymes, elongation factors, splicing factors, 3'-end-processing complexes, and termination factors. These transitions are coordinated by the dynamic phosphorylation of the C-terminal domain (CTD) of the largest subunit of RNAPII (Rpb1). The CTD is composed of reiterated heptapeptide repeats (Y(1)S(2)P(3)T(4)S(5)P(6)S(7)) that undergo phosphorylation and dephosphorylation as RNAPII transitions through the transcription cycle. An essential phosphatase in this process is Ssu72, which exhibits catalytic specificity for Ser(P)(5) and Ser(P)(7). Ssu72 is unique in that it is specific for Ser(P)(5) in one orientation of the CTD and for Ser(P)(7) when bound in the opposite orientation. Moreover, Ssu72 interacts with components of the initiation machinery and affects start site selection yet is an integral component of the CPF 3'-end-processing complex. Here we provide a comprehensive view of the effects of Ssu72 with respect to its Ser(P)(5) phosphatase activity. We demonstrate that Ssu72 dephosphorylates Ser(P)(5) at the initiation-elongation transition. Furthermore, Ssu72 indirectly affects the levels of Ser(P)(2) during the elongation stage of transcription but does so independent of its catalytic activity.

Cap Completion and C-Terminal Repeat Domain Kinase Recruitment Underlie the Initiation-Elongation Transition of RNA Polymerase II

After transcription initiation, RNA polymerase (Pol) II escapes from the promoter and recruits elongation factors. The molecular basis for the initiation-elongation factor exchange during this transition remains poorly understood. Here, we used chromatin immunoprecipitation (ChIP) to elucidate the initiation-elongation transition of Pol II in the budding yeast Saccharomyces cerevisiae. We show that the early Pol II elongation factor Spt5 contributes to stable recruitment of the mRNA capping enzymes Cet1, Ceg1, and Abd1. Genome-wide occupancy for Cet1 and Ceg1 is restricted to the transcription start site (TSS), whereas occupancy for Abd1 peaks at ~110 nucleotides downstream, and occupancy for the cap-binding complex (CBC) rises subsequently. Abd1 and CBC are important for recruitment of the kinases Ctk1 and Bur1, which promote elongation and capping enzyme release. These results suggest that cap completion stimulates productive Pol II elongation.

Dynamic Architecture of a Minimal RNA Polymerase II Open Promoter Complex

The open promoter complex (OC) is a central intermediate during transcription initiation that contains a DNA bubble. Here, we employ single-molecule Förster resonance energy transfer experiments and Nano-Positioning System analysis to determine the three-dimensional architecture ofa minimal OC consisting of promoter DNA, including a TATA box and an 11-nucleotide mismatched region around the transcription start site, TATA box-binding protein (TBP), RNA polymerase (Pol) II, and general transcription factor (TF)IIB and TFIIF. In this minimal OC, TATA-DNA and TBP reside above the Pol II cleft between clamp and protrusion domains. Downstream DNA is dynamically loaded into and unloaded from the Pol II cleft at a timescale of seconds. The TFIIB core domain is displaced from the Pol II wall, where it is located in the closed promoter complex. These results reveal large overall structural changes during the initiation-elongation transition, which are apparently accommodated by the intrinsic flexibility of TFIIB.

Dynamic Architecture of the RNA Polymerase II Open Promoter Complex

Transcription initiation of eukaryotic protein-coding genes is a multi-step process that requires assembly of RNA polymerase (Pol) II and the general transcription factors into the closed complex. Then, the DNA surrounding the TSS is melted and inserted into the Pol II active center cleft to form the open complex (OC), the starting point for RNA synthesis. The OC is hence a central intermediate in the initiation-elongation transition and insights into its structure are crucial for understanding the molecular mechanism of transcription initiation.X-ray crystallography has provided detailed insights into the architecture of Pol II in different functional states, but structural studies of transcription initiation have been hindered by the flexibility of Pol II initiation complexes. As a consequence, the structure of the OC remains unknown.Here, we employ single-molecule Forster Resonance Energy Transfer (smFRET) and Nano-Positioning System (NPS)analysis [1] to determine the 3-dimensional architecture of a minimal OC consisting of Pol II, promoter DNA, TBP, TFIIB, and -IIF. Briefly, by using smFRET, we measure distances within double labeled OCs and the NPS computes 3-dimensional position probability densities of unknown sites on upstream DNA, TATA-DNA, TBP, and TFIIB, which allows us to build a model of the OC.We show that in the OC, TBP and TATA-DNA reside above the Pol II cleft between clamp and protrusion. Downstream DNA is dynamically loaded into the cleft and unloaded from the cleft at a timescale of seconds. The TFIIB core domain is displaced from the Pol II wall, where it was located in the closed complex. Hence, our results define the overall structural changes during the initiation-elongation transition, and reveal a key role for the intrinsic flexibility of TFIIB in accommodating these changes.[1] Muschielok et al., Nature Methods, 5, 965 (2008).

Nano positioning system reveals the course of upstream and nontemplate DNA within the RNA polymerase II elongation complex

Crystallographic studies of the RNA polymerase II (Pol II) elongation complex (EC) revealed the locations of downstream DNA and the DNA-RNA hybrid, but not the course of the nontemplate DNA strand in the transcription bubble and the upstream DNA duplex. Here we used single-molecule Fluorescence Resonance Energy Transfer (smFRET) experiments to locate nontemplate and upstream DNA with our recently developed Nano Positioning System (NPS). In the resulting complete model of the Pol II EC, separation of the nontemplate from the template strand at position +2 involves interaction with fork loop 2. The nontemplate strand passes loop β10-β11 on the Pol II lobe, and then turns to the other side of the cleft above the rudder. The upstream DNA duplex exits at an approximately right angle from the incoming downstream DNA, and emanates from the cleft between the protrusion and clamp. Comparison with published data suggests that the architecture of the complete EC is conserved from bacteria to eukaryotes and that upstream DNA is relocated during the initiation–elongation transition.

Monitoring RNA transcription in real time by using surface plasmon resonance

The decision to elongate or terminate the RNA chain at specific DNA template positions during transcription is kinetically regulated, but the methods used to measure the rates of these processes have not been sufficiently quantitative to permit detailed mechanistic analysis of the steps involved. Here, we use surface plasmon resonance (SPR) technology to monitor RNA transcription by Escherichia coli RNA polymerase (RNAP) in solution and in real time. We show that binding of RNAP to immobilized DNA templates to form active initiation or elongation complexes can be resolved and monitored by this method, and that changes during transcription that involve the gain or loss of bound mass, including the release of the sigma factor during the initiation-elongation transition, the synthesis of the RNA transcript, and the release of core RNAP and nascent RNA at intrinsic terminators, can all be observed. The SPR method also permits the discrimination of released termination products from paused and other intermediate complexes at terminators. We have used this approach to show that the rate constant for transcript release at intrinsic terminators tR2 and tR' is approximately 2-3 s(-1) and that the extent of release at these terminators is consistent with known termination efficiencies. Simulation techniques have been used to fit the measured parameters to a simple kinetic model of transcription and the implications of these results for transcriptional regulation are discussed.

Promoter Escape by Escherichia coli RNA Polymerase.

Promoter escape is the process that an initiated RNA polymerase (RNAP) molecule undergoes to achieve the initiation-elongation transition. Having made this transition, an RNAP molecule would be relinquished from its promoter hold to perform productive (full-length) transcription. Prior to the transition, this process is accompanied by abortive RNA formation-the amount and pattern of which is controlled by the promoter sequence information. Qualitative and quantitative analysis of abortive/productive transcription from several Escherichia coli promoters and their sequence variants led to the understanding that a strong (RNAP-binding) promoter is more likely to be rate limited (during transcription initiation) at the escape step and produce abortive transcripts. Of the two subelements in a promoter, the PRR (the core Promoter Recognition Region) was found to set the initiation frequency and the rate-limiting step, while the ITS (the Initial Transcribed Sequence region) modulated the ratio of abortive versus productive transcription. The highly abortive behavior of E. coli RNAP could be ameliorated by the presence of Gre (transcript cleavage stimulatory) factor(s), linking the first step in abortive RNA formation by the initial transcribing complexes (ITC) to RNAP backtracking. The discovery that translocation during the initiation stage occurs via DNA scrunching provided the source of energy that converts each ITC into a highly unstable "stressed intermediate." Mapping all of the biochemical information onto an X-ray crystallographic structural model of an open complex gave rise to a plausible mechanism of transcription initiation. The chapter concludes with contemplations of the kinetics and thermodynamics of abortive initiation-promoter escape.

The initiation-elongation transition: lateral mobility of RNA in RNA polymerase II complexes is greatly reduced at +8/+9 and absent by +23.

RNA polymerase II transcription complexes stalled shortly after initiation over a repetitive segment of the template can undergo efficient transcript slippage, during which the 3' end of the RNA slides upstream and then re-pairs with the template, allowing transcription to continue. In the present study, we have used transcript slippage as an assay to identify possible structural transitions that occur as the polymerase passes from the initiation to the elongation phase of transcription. We reasoned that transcript slippage would not occur in fully processive complexes. We constructed a series of templates that allowed us to stall RNA polymerase II after the synthesis of a repetitive sequence (5'-CUCUCU-3') at varying distances downstream of +1. We found that polymerase must synthesize at least a 23-nt RNA to attain resistance to transcript slippage. The ability to undergo slippage was lost in two discrete steps, suggestive of two distinct transitions. The first transition is the formation of the 8- to 9-bp mature RNA-DNA hybrid, when slippage abruptly dropped by 10-fold. However, easily detectable slippage continued until 14 more bonds were made. Thus, although the transcript becomes tightly constrained within the transcription complex once the hybrid reaches its final length, much more RNA synthesis is required before the RNA is no longer able to slip upstream along the template. This last point may reflect an important stabilizing role for the interaction of the polymerase with the transcript well upstream of the RNA-DNA hybrid.