Steps Of Transcription Initiation Research Articles

Structures illustrate step-by-step mitochondrial transcription initiation

Transcription initiation is a key regulatory step in gene expression during which RNA polymerase (RNAP) initiates RNA synthesis de novo, and the synthesized RNA at a specific length triggers the transition to the elongation phase. Mitochondria recruit a single-subunit RNAP and one or two auxiliary factors to initiate transcription. Previous studies have revealed the molecular architectures of yeast1 and human2 mitochondrial RNAP initiation complexes (ICs). Here we provide a comprehensive, stepwise mechanism of transcription initiation by solving high-resolution cryogenic electron microscopy (cryo-EM) structures of yeast mitochondrial RNAP and the transcription factor Mtf1 catalysing two- to eight-nucleotide RNA synthesis at single-nucleotide addition steps. The growing RNA–DNA is accommodated in the polymerase cleft by template scrunching and non-template reorganization, creating stressed intermediates. During early initiation, non-template strand scrunching and unscrunching destabilize the short two- and three-nucleotide RNAs, triggering abortive synthesis. Subsequently, the non-template reorganizes into a base-stacked staircase-like structure supporting processive five- to eight-nucleotide RNA synthesis. The expanded non-template staircase and highly scrunched template in IC8 destabilize the promoter interactions with Mtf1 to facilitate initiation bubble collapse and promoter escape for the transition from initiation to the elongation complex (EC). The series of transcription initiation steps, each guided by the interplay of multiple structural components, reveal a finely tuned mechanism for potential regulatory control.

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.

RNA-Mediated Feedback Control of Transcriptional Condensates

Regulation of biological processes typically incorporates mechanisms that initiate and terminate the process and, where understood, these mechanisms often involve feedback control. Regulation of transcription is a fundamental cellular process where the mechanisms involved in initiation have been studied extensively, but those involved in arresting the process are poorly understood. Modeling of the potential roles of RNA in transcriptional control suggested a non-equilibrium feedback control mechanism where low levels of RNA promote condensates formed by electrostatic interactions whereas relatively high levels promote dissolution of these condensates. Evidence from invitro and invivo experiments support a model where RNAs produced during early steps in transcription initiation stimulate condensate formation, whereas the burst of RNAs produced during elongation stimulate condensate dissolution. We propose that transcriptional regulation incorporates a feedback mechanism whereby transcribed RNAs initially stimulate but then ultimately arrest the process.

Cryo-EM Structures Reveal Transcription Initiation Steps by Yeast Mitochondrial RNA Polymerase

Mitochondrial RNA polymerase (mtRNAP) is crucial in cellular energy production, yet understanding of mitochondrial DNA transcription initiation lags that of bacterial and nuclear DNA transcription. We report structures of two transcription initiation intermediate states of yeast mtRNAP that explain promoter melting, template alignment, DNA scrunching, abortive synthesis, and transition into elongation. In the partially melted initiation complex (PmIC), transcription factor MTF1 makes base-specific interactions with flipped non-template (NT) nucleotides "AAGT" at -4 to -1 positions of the DNA promoter. In the initiation complex (IC), the template in the expanded 7-mer bubble positions the RNA and NTP analog UTPαS, while NT scrunches into an NT loop. The scrunched NT loop is stabilized by the centrally positioned MTF1 C-tail. The IC and PmIC states coexist in solution, revealing a dynamic equilibrium between two functional states. Frequent scrunching/unscruching transitions and the imminent steric clashes of the inflating NT loop and growing RNA:DNA with the C-tail explain abortive synthesis and transition into elongation.

ELL Facilitates RNA Polymerase II–Mediated Transcription of Human Epidermal Proliferation Genes

The first step of mRNA synthesis begins with the recruitment and stabilization of RNA polymerase II (Pol II) and other components of the transcription apparatus to the promoter and formation of a preinitiation complex. This step of transcription initiation has been considered the rate-limiting step and controls expression of a significant number of genes. Recent evidence has also demonstrated that transcriptional control at the postinitiation steps, including promoter proximal pausing and elongation, are central for regulating gene expression (Adelman and Lis, 2012).

The nuclear cap-binding complex as choreographer of gene transcription and pre-mRNA processing.

The largely nuclear cap-binding complex (CBC) binds to the 5' caps of RNA polymerase II (RNAPII)-synthesized transcripts and serves as a dynamic interaction platform for a myriad of RNA processing factors that regulate gene expression. While influence of the CBC can extend into the cytoplasm, here we review the roles of the CBC in the nucleus, with a focus on protein-coding genes. We discuss differences between CBC function in yeast and mammals, covering the steps of transcription initiation, release of RNAPII from pausing, transcription elongation, cotranscriptional pre-mRNA splicing, transcription termination, and consequences of spurious transcription. We describe parameters known to control the binding of generic or gene-specific cofactors that regulate CBC activities depending on the process(es) targeted, illustrating how the CBC is an ever-changing choreographer of gene expression.

The σ Subunit-Remodeling Factors: An Emerging Paradigms of Transcription Regulation.

Transcription initiation is a key checkpoint and highly regulated step of gene expression. The sigma (σ) subunit of RNA polymerase (RNAP) controls all transcription initiation steps, from recognition of the −10/−35 promoter elements, upon formation of the closed promoter complex (RPc), to stabilization of the open promoter complex (RPo) and stimulation of the primary steps in RNA synthesis. The canonical mechanism to regulate σ activity upon transcription initiation relies on activators that recognize specific DNA motifs and recruit RNAP to promoters. This mini-review describes an emerging group of transcriptional regulators that form a complex with σ or/and RNAP prior to promoter binding, remodel the σ subunit conformation, and thus modify RNAP activity. Such strategy is widely used by bacteriophages to appropriate the host RNAP. Recent findings on RNAP-binding protein A (RbpA) from Mycobacterium tuberculosis and Crl from Escherichia coli suggest that activator-driven changes in σ conformation can be a widespread regulatory mechanism in bacteria.

The Role of Transcription Factor - DNA Residence Time in Transcription

Transcription is regulated by the interplay of many different factors in a sequence of intrinsically stochastic processes. For most genes, this leads to short periods of activity (called bursts) followed by quiescence. Binding of transcription factors (TFs), either activators or repressors, represents the first step of transcription initiation. Fluorescence microscopy revealed that most TFs bind dynamically to DNA while each possesses an unique residence time. How this DNA residence time influences the activity of the TF remains unclear. We probe the influence of TF DNA residence time on transcription by generating TFs that target a certain gene locus and only differ in their DNA residence times. Therefore, we designed series of transcription activator-like effectors (TALEs) based TFs with varying numbers of nucleotide-recognizing repeat domains targeting the promoter region of a reporter gene. We characterized the DNA residence times of our TALE-TFs by single molecule tracking experiments in living cells. To determine the transcriptional output and frequency of bursts, we use single molecule RNA fluorescence in situ hybridization (smRNA-FISH). For a repressing system, a factor of six in DNA residence time accounts for a decrease of 50% in transcript level, by down regulation of burst frequency at constant burst size. Similarly, different DNA binding domains differentially affect transcription output in an activating system. Together our experiments demonstrate that the DNA residence time of TFs is a regulatory factor of transcription.

Cryo-EM Structures Reveal Transcription Initiation Steps by Yeast Mitochondrial RNA Polymerase

Understanding of mitochondrial DNA transcription initiation lags that of bacterial and nuclear DNA transcription. We have solved the structures of yeast mitochondrial RNA polymerase transcription pre-initiation complex (PIC) and initiation complex (IC) with fully resolved transcription bubbles. The structures explain promoter melting, template alignment, DNA scrunching, transition into elongation, and abortive synthesis. Partially melted DNA in PIC is caused by interactions of MTF1 with four flipped non-template bases. In IC, the bubble expands, a largescale movement aligns the template with RNA/NTP in the active site, and the downstream DNA is repositioned. The IC-state captures the scrunched DNA as an NT-loop that interacts with the centrally positioned MTF1 C-tail. Coexisting scrunched and unscrunched states and imminent steric clashes of NT-loop and RNA:DNA with C-tail explain abortive synthesis and transition into elongation. The IC-state with UTPαS poised for catalytic incorporation provides a platform to assess the toxicity of antiviral nucleosides/nucleotides like remdesivir.

Transcription closed and open complex formation coordinate expression of genes with a shared promoter region.

Many genes are spaced closely, allowing coordination without explicit control through shared regulatory elements and molecular interactions. We study the dynamics of a stochastic model of a gene-pair in a head-to-head configuration, sharing promoter elements, which accounts for the rate-limiting steps in transcription initiation. We find that only in specific regions of the parameter space of the rate-limiting steps is orderly coexpression exhibited, suggesting that successful cooperation between closely spaced genes requires the coevolution of compatible rate-limiting step configuration. The model predictions are validated using in vivo single-cell, single-RNA measurements of the dynamics of pairs of genes sharing promoter elements. Our results suggest that, in E. coli, the kinetics of the rate-limiting steps in active transcription can play a central role in shaping the dynamics of gene-pairs sharing promoter elements.

A negative feedback mechanism links UBC gene expression to ubiquitin levels by affecting RNA splicing rather than transcription

UBC gene plays a critical role in maintaining ubiquitin (Ub) homeostasis. It is upregulated under stress conditions, and herein we report that it is downregulated upon Ub overexpression. Downregulation occurs in a dose-dependent manner, suggesting the existence of a fine-tuned Ub sensing mechanism. This “sensor” requires a conjugation competent ubiquitin to detect Ub levels. Searching the sensor among the transcription factors involved in basal and stress-induced UBC gene expression was unsuccessful. Neither HSF1 and HSF2, nor Sp1 and YY1 are affected by the increased Ub levels. Moreover, mutagenesis of their binding sites in the UBC promoter-driven reporter constructs does not impair the downmodulation effect. Epigenetic studies show that H2A and H2B ubiquitination within the UBC promoter region is unchanged upon ubiquitin overexpression. Noteworthy, quantification of nascent RNA molecules excludes that the downmodulation arises in the transcription initiation step, rather pointing towards a post-transcriptional mechanism. Indeed, a significantly higher fraction of unspliced UBC mRNA is detected in ubiquitin overexpressing cells, compared to empty vector transfected cells. Our findings suggest how increasing cellular ubiquitin levels may control the expression of UBC gene by negatively affecting the splicing of its pre-mRNA, providing a straightforward feedback strategy for the homeostatic control of ubiquitin pools.

Regulation of asymmetries in the kinetics and protein numbers of bacterial gene expression

Genetic circuits change the status quo of cellular processes when their protein numbers cross thresholds. We investigate the regulation of RNA and protein threshold crossing propensities in Escherichia coli. From in vivo single RNA time-lapse microscopy data from multiple promoters, mutants, induction schemes and media, we study the asymmetry and tailedness (quantified by the skewness and kurtosis, respectively) of the distributions of time intervals between transcription events. We find that higher thresholds can be reached by increasing the skewness and kurtosis, which is shown to be achievable without affecting mean and coefficient of variation, by regulating the rate-limiting steps in transcription initiation. Also, they propagate to the skewness and kurtosis of the distributions of protein expression levels in cell populations. The results suggest that the asymmetry and tailedness of RNA and protein numbers in cell populations, by controlling the propensity for threshold crossing, and due to being sequence dependent and subject to regulation, may be key regulatory variables of decision-making processes in E. coli.

Region 3.2 of the σ factor controls the stability of rRNA promoter complexes and potentiates their repression by DksA.

The σ factor drives promoter recognition by bacterial RNA polymerase (RNAP) and is also essential for later steps of transcription initiation, including RNA priming and promoter escape. Conserved region 3.2 of the primary σ factor (‘σ finger’) directly contacts the template DNA strand in the open promoter complex and facilitates initiating NTP binding in the active center of RNAP. Ribosomal RNA promoters are responsible for most RNA synthesis during exponential growth but should be silenced during the stationary phase to save cell resources. In Escherichia coli, the silencing mainly results from the action of the secondary channel factor DksA, which together with ppGpp binds RNAP and dramatically decreases the stability of intrinsically unstable rRNA promoter complexes. We demonstrate that this switch depends on the σ finger that destabilizes RNAP–promoter interactions. Mutations in the σ finger moderately decrease initiating NTP binding but significantly increase promoter complex stability and reduce DksA affinity to the RNAP–rRNA promoter complex, thus making rRNA transcription less sensitive to DksA/ppGpp both in vitro and in vivo. Thus, destabilization of rRNA promoter complexes by the σ finger makes them a target for robust regulation by the stringent response factors under stress conditions.

ChIP-exo interrogation of Crp, DNA, and RNAP holoenzyme interactions.

Numerous in vitro studies have yielded a refined picture of the structural and molecular associations between Cyclic-AMP receptor protein (Crp), the DNA motif, and RNA polymerase (RNAP) holoenzyme. In this study, high-resolution ChIP-exonuclease (ChIP-exo) was applied to study Crp binding in vivo and at genome-scale. Surprisingly, Crp was found to provide little to no protection of the DNA motif under activating conditions. Instead, Crp demonstrated binding patterns that closely resembled those generated by σ70. The binding patterns of both Crp and σ70 are indicative of RNAP holoenzyme DNA footprinting profiles associated with stages during transcription initiation that occur post-recruitment. This is marked by a pronounced advancement of the template strand footprint profile to the +20 position relative to the transcription start site and a multimodal distribution on the nontemplate strand. This trend was also observed in the familial transcription factor, Fnr, but full protection of the motif was seen in the repressor ArcA. Given the time-scale of ChIP studies and that the rate-limiting step in transcription initiation is typically post recruitment, we propose a hypothesis where Crp is absent from the DNA motif but remains associated with RNAP holoenzyme post-recruitment during transcription initiation. The release of Crp from the DNA motif may be a result of energetic changes that occur as RNAP holoenzyme traverses the various stable intermediates towards elongation complex formation.

XPD-The Lynchpin of NER: Molecule, Gene, Polymorphisms, and Role in Colorectal Carcinogenesis.

In mammals the bulky DNA adduct lesions known to result in deleterious phenotypes are acted upon and removed from the genomic DNA by nucleotide excision repair (NER) pathway. TFIIH multi-protein complex with its important helicase–Xeroderma Pigmentosum Protein (XPD) serves as the pivotal factor for opening up of the damaged lesion DNA site and carry out the repair process. The initial damage verification step of the TFIIH is in part dependent upon the helicase activity of XPD. Besides, XPD is also actively involved in the initiation steps of transcription and in the regulation of the cell cycle and apoptosis. In this review, we will be exploring the new insights in scientific research on the functioning of the NER pathway, the role of TFIIH as the central complex of NER, the pivotal helicase XPD as the lynchpin of NER and the effects of various single nucleotide polymorphisms (SNPs) of XPD on its functioning and their consequent role in colorectal carcinogenesis.

Investigation of specific interactions between T7 promoter and T7 RNA polymerase by force spectroscopy using atomic force microscope.

The specific recognition and binding of promoter and RNA polymerase is the first step of transcription initiation in bacteria and largely determines transcription activity. Therefore, direct analysis of the interaction between promoter and RNA polymerase in vitro may be a new strategy for promoter characterization, to avoid interference due to the cell's biophysical condition and other regulatory elements. In the present study, the specific interaction between T7 promoter and T7 RNA polymerase was studied as a model system using force spectroscopy based on atomic force microscope (AFM). The specific interaction between T7 promoter and T7 RNA polymerase was verified by control experiments, and the rupture force in this system was measured as 307.2 ± 6.7 pN. The binding between T7 promoter mutants with various promoter activities and T7 RNA polymerase was analyzed. Interaction information including rupture force, rupture distance and binding percentage were obtained in vitro, and reporter gene expression regulated by these promoters was also measured according to a traditional promoter activity characterization method in vivo Using correlation analysis, it was found that the promoter strength characterized by reporter gene expression was closely correlated with rupture force and the binding percentage by force spectroscopy. These results indicated that the analysis of the interaction between promoter and RNA polymerase using AFM-based force spectroscopy was an effective and valid approach for the quantitative characterization of promoters.

Conformational heterogeneity and bubble dynamics in single bacterial transcription initiation complexes.

Transcription initiation is a major step in gene regulation for all organisms. In bacteria, the promoter DNA is first recognized by RNA polymerase (RNAP) to yield an initial closed complex. This complex subsequently undergoes conformational changes resulting in DNA strand separation to form a transcription bubble and an RNAP-promoter open complex; however, the series and sequence of conformational changes, and the factors that influence them are unclear. To address the conformational landscape and transitions in transcription initiation, we applied single-molecule Förster resonance energy transfer (smFRET) on immobilized Escherichia coli transcription open complexes. Our results revealed the existence of two stable states within RNAP–DNA complexes in which the promoter DNA appears to adopt closed and partially open conformations, and we observed large-scale transitions in which the transcription bubble fluctuated between open and closed states; these transitions, which occur roughly on the 0.1 s timescale, are distinct from the millisecond-timescale dynamics previously observed within diffusing open complexes. Mutational studies indicated that the σ70 region 3.2 of the RNAP significantly affected the bubble dynamics. Our results have implications for many steps of transcription initiation, and support a bend-load-open model for the sequence of transitions leading to bubble opening during open complex formation.

How to switch the motor on: RNA polymerase initiation steps at the single‐molecule level

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 Förster 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.

Effects of σ factor competition are promoter initiation kinetics dependent

In Escherichia coli, the expression of a σ factor is expected to indirectly down-regulate the expression of genes recognized by another σ factor, due to σ factor competition for a limited pool of RNA polymerase core enzymes. Evidence suggests that the sensitivity of genes to indirect down-regulation differs widely. We studied the variability in this sensitivity in promoters primarily recognized by RNAP holoenzymes carrying σ70. From qPCR and live single-cell, single-RNA measurements of the transcription kinetics of several σ70-dependent promoters in various conditions and from the analysis of σ factors population-dependent models of transcription initiation, we find that, the smaller is the time-scale of the closed complex formation relative to the open complex formation, the weaker is a promoter's responsiveness to changes in σ38 numbers. We conclude that, in E. coli, a promoter's responsiveness to indirect regulation by σ factor competition is determined by the sequence-dependent kinetics of the rate limiting steps of transcription initiation.

Dissecting the stochastic transcription initiation process in live Escherichia coli.

We investigate the hypothesis that, in Escherichia coli, while the concentration of RNA polymerases differs in different growth conditions, the fraction of RNA polymerases free for transcription remains approximately constant within a certain range of these conditions. After establishing this, we apply a standard model-fitting procedure to fully characterize the in vivo kinetics of the rate-limiting steps in transcription initiation of the Plac/ara-1 promoter from distributions of intervals between transcription events in cells with different RNA polymerase concentrations. We find that, under full induction, the closed complex lasts ∼788 s while subsequent steps last ∼193 s, on average. We then establish that the closed complex formation usually occurs multiple times prior to each successful initiation event. Furthermore, the promoter intermittently switches to an inactive state that, on average, lasts ∼87 s. This is shown to arise from the intermittent repression of the promoter by LacI. The methods employed here should be of use to resolve the rate-limiting steps governing the in vivo dynamics of initiation of prokaryotic promoters, similar to established steady-state assays to resolve the in vitro dynamics.