Translocation Of RNA Polymerase Research Articles

Force and the α-C-terminal domains bias RNA polymerase recycling

After an RNA polymerase reaches a terminator, instead of dissociating from the template, it may diffuse along the DNA and recommence RNA synthesis from the previous or a different promoter. Magnetic tweezers were used to monitor such secondary transcription and determine the effects of low forces assisting or opposing translocation, protein roadblocks, and transcription factors. Remarkably, up to 50% of Escherichia coli (E. coli) RNA polymerases diffused along the DNA after termination. Force biased the direction of diffusion (sliding) and the velocity increased rapidly with force up to 0.7 pN and much more slowly thereafter. Sigma factor 70 (σ70) likely remained associated with the DNA promoting sliding and enabling re-initiation from promoters in either orientation. However, deletions of the α-C-terminal domains severely limited the ability of RNAP to turn around between successive rounds of transcription. The addition of elongation factor NusG, which competes with σ70 for binding to RNAP, limited additional rounds of transcription. Surprisingly, sliding RNA polymerases blocked by a DNA-bound lac repressor could slowly re-initiate transcription and were not affected by NusG, suggesting a σ-independent pathway. Low forces effectively biased promoter selection suggesting a prominent role for topological entanglements that affect RNA polymerase translocation.

Nucleosomes play a dual role in regulating transcription dynamics

Transcription has a mechanical component, as the translocation of the transcription machinery or RNA polymerase (RNAP) on DNA or chromatin is dynamically coupled to the chromatin torsion. This posits chromatin mechanics as a possible regulator of eukaryotic transcription, however, the modes and mechanisms of this regulation are elusive. Here, we first take a statistical mechanics approach to model the torsional response of topology-constrained chromatin. Our model recapitulates the experimentally observed weaker torsional stiffness of chromatin compared to bare DNA and proposes structural transitions of nucleosomes into chirally distinct states as the driver of the contrasting torsional mechanics. Coupling chromatin mechanics with RNAP translocation in stochastic simulations, we reveal a complex interplay of DNA supercoiling and nucleosome dynamics in governing RNAP velocity. Nucleosomes play a dual role in controlling the transcription dynamics. The steric barrier aspect of nucleosomes in the gene body counteracts transcription via hindering RNAP motion, whereas the chiral transitions facilitate RNAP motion via driving a low restoring torque upon twisting the DNA. While nucleosomes with low dissociation rates are typically transcriptionally repressive, highly dynamic nucleosomes offer less of a steric barrier and enhance the transcription elongation dynamics of weakly transcribed genes via buffering DNA twist. We use the model to predict transcription-dependent levels of DNA supercoiling in segments of the budding yeast genome that are in accord with available experimental data. The model unveils a paradigm of DNA supercoiling-mediated interaction between genes and makes testable predictions that will guide experimental design.

RNA polymerase is a unique Maxwell’s demon that converts its transcribing genetic information to free energy for its movement

RNA polymerase (RNAP) catalyzes RNA synthesis from template DNA via translocation on the DNA. The experimental value of the free energy change required for RNAP translocation exhibits an unexplainable discrepancy. To address this, we propose a transcription system model based on information thermodynamics. The state function of RNAP was defined from its position on the template DNA (m), its migration direction (d), and the deoxyribonucleotide (dNTP) that it transcribes (N). Based on the state function, the free energy change in the RNAP translocation was defined as the sum of ΔGd in the movement fluctuation or the mutual entropy term kBTlogP(N) from the appearance probability P(N) of N to be transcribed by RNAP. In conclusion, a discrepancy in free energy change values is due to either ΔGd or kBTlogP(N) involvement. The discrepancy highlights that RNAP is a unique information-work converter or Maxwell’s demon that can feed back the obtained dNTP-type information into its self-movement along the DNA.

A spatially resolved stochastic model reveals the role of supercoiling in transcription regulation.

In Escherichia coli, translocation of RNA polymerase (RNAP) during transcription introduces supercoiling to DNA, which influences the initiation and elongation behaviors of RNAP. To quantify the role of supercoiling in transcription regulation, we developed a spatially resolved supercoiling model of transcription. The integrated model describes how RNAP activity feeds back with the local DNA supercoiling and how this mechanochemical feedback controls transcription, subject to topoisomerase activities and stochastic topological domain formation. This model establishes that transcription-induced supercoiling mediates the cooperation of co-transcribing RNAP molecules in highly expressed genes, and this cooperation is achieved under moderate supercoiling diffusion and high topoisomerase unbinding rates. It predicts that a topological domain could serve as a transcription regulator, generating substantial transcriptional noise. It also shows the relative orientation of two closely arranged genes plays an important role in regulating their transcription. The model provides a quantitative platform for investigating how genome organization impacts transcription.

NusG controls transcription pausing and RNA polymerase translocation throughout the Bacillus subtilis genome

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.

Stringent control of the RNA-dependent RNA polymerase translocation revealed by multiple intermediate structures

Each polymerase nucleotide addition cycle is associated with two primary conformational changes of the catalytic complex: the pre-chemistry active site closure and post-chemistry translocation. While active site closure is well interpreted by numerous crystallographic snapshots, translocation intermediates are rarely captured. Here we report three types of intermediate structures in an RNA-dependent RNA polymerase (RdRP). The first two types, captured in forward and reverse translocation events, both highlight the role of RdRP-unique motif G in restricting the RNA template movement, corresponding to the rate-limiting step in translocation. By mutating two critical residues in motif G, we obtain the third type of intermediates that may mimic the transition state of this rate-limiting step, demonstrating a previously unidentified movement of the template strand. We propose that a similar strategy may be utilized by other classes of nucleic acid polymerases to ensure templating nucleotide positioning for efficient catalysis through restricting interactions with template RNA.

Abstract A72: Novel approach for upregulation of endogenous immunostimulatory targets for cancer therapy

Abstract Small activating RNAs (saRNAs) are short, double-stranded oligonucleotides designed to specifically upregulate target mRNA and protein expression by loading into an Ago2 complex for nuclear translocation and activation of RNA polymerase II activity. We have developed MTL-CEBPA, an saRNA-based therapeutic designed to increase the expression of the CEBPA transcription factor that acts as both a tumor suppressor and a key modulator of the tumor microenvironment. In preclinical tumor models MTL-CEBPA has single-agent antitumor activity and very significantly improves the activity of both sorafenib and checkpoint inhibitors through impacting the immunosuppressive tumor microenvironment. This is the first saRNA drug to enter clinical trials, and we have been investigating whether this technology could be extended to activate endogenous immune stimulatory genes for utilization in cancer immunotherapy. We designed saRNA to target the IL-23A, IL-36g, OX40L, and UCP2 transcripts using our proprietary algorithm that bind to long noncoding RNA at position +2000 to -2000 nucleotides relative to the transcriptional start site. At least five 19-nucleotide sequences from the algorithm for each target were synthesized with mUmU overhangs at the 3´ ends of both strands to improve stability and reduce nonspecific immunogenicity. The saRNAs were screened by transfection in human (HepG2 and A549) or murine (CT26, BNL.1ME and RAW264.7) cells, and mRNA levels of the target genes were measured by qPCR. The screening process identified active saRNAs for all 4 targets leading to upregulation of target mRNA by 1.9 to 6.7-fold at 48-72 hours post transfection compared to oligonucleotide transfection controls. Sequence-dependent specificity was confirmed by modifying 2 to 3 nucleotide residues in the seed binding region of the lead sequences, which in each case significantly reduced induction of target mRNA compared to the parental saRNA sequences. For each lead saRNA, increased target protein was also confirmed either by immunofluorescence or ELISA and increased functional activity was confirmed either by impact on downstream targets or phenotypic activity. For example, overexpression of UCP2 transgene in tumor cell lines has been demonstrated by others to suppress tumor cell proliferation and imitate an antitumor immune cycle. We showed that these effects were recapitulated in vitro by increasing endogenous UCP2 expression using saRNA. Here, we demonstrate the utilization of saRNA technology to upregulate immune stimulatory targets for immunotherapy. We are currently progressing efficacy studies in a number of preclinical tumor models. Citation Format: Choon Ping Tan, Laura Sinigaglia, Siv A Hegre, Alexandre Debacker, Albert Kwok, Pal Saetrom, Matthew Catley, David C. Blakey, Nagy Habib. Novel approach for upregulation of endogenous immunostimulatory targets for cancer therapy [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr A72.

Fractional‐nucleotide translocation in sequence‐dependent pausing by RNA polymerase: Single‐molecule picometer‐resolution nanopore tweezers (SPRNT)

Single‐molecule picometer‐resolution nanopore tweezers (SPRNT) enables monitoring of translocation of a nucleic‐acid motor protein on a nucleic‐acid track with sequence registration, sub‐nucleotide spatial resolution, sub‐millisecond temporal resolution, and the ability to apply assisting and opposing forces.We have demonstrated the ability of SPRNT to monitor translocation of RNA polymerase (RNAP) relative to the DNA template strand, the DNA non‐template strand, and the RNA product strand in transcription, and we have applied SPRNT to analyze RNAP translocation in transcription elongation and sequence‐dependent transcriptional pausing.The results show that sequence‐dependent transcriptional pausing involves a “half‐translocated” state in which RNAP translocates relative to the DNA template strand by 0.5 nucleotides. The “half‐translocated” state is unable to bind the next NTP substrate, presumably because RNAP translocation by 0.5 nt does not align the next template‐strand nucleotide with the RNAP active‐center NTP addition site. The half‐translocated state interconverts rapidly (~0.1–1 s) with the “pre‐translocated” state and converts slowly (~1 s) to the NTP‐binding‐competent “post‐translocated state.” In the presence of saturating NTPs, conversion to the post‐translocated state is followed by pause escape. In the presence of limiting NTPs, conversion to the post‐translocated state is followed by return to interconverting half‐translocated and pre‐translocated states and continued pausing.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Transcription Machine Studied in a Nutshell on T7 RNA Polymerase Mechanochemistry, Fidelity Control, and Bursting Activity

RNA polymerase (RNAP) is an essential enzyme that transcribes DNA into messenger RNA to further express proteins. Bacteriophage T7 RNAP is widely utilized in lab gene expression system and synthetic circuit as it transcribes self-sufficiently, without transcription factors, and with high promoter activity. We have investigated T7 RNAP on both its mechano-chemical couplings and fidelity control during transcription, combining physical modeling with structural dynamics simulations. In particular, we have implemented extensive atomistic molecular dynamics (MD) simulations and constructed the Markov state model (MSM) to investigate product release and translocation process of T7 RNAP, as it couples Brownian thermal motions with essential structural elements to assist PPi release, to facilitate the RNAP translocation, and to prevent backtracking. Meanwhile, we systematically quantified the nucleotide selectivity of this non-proofreading RNAP at multiple kinetic checkpoints prior to the full nucleotide incorporation. Essential selection against the non-cognate nucleotide species appears to happen at early checkpoints at the pre-insertion and right after, with a critical amino acid serving as a paw for selective ratcheting RNAP along the DNA. In addition, we also model the complete transcription process along with RNAP traffic, as positive supercoiling is accumulated in front of a leading RNAP to inhibit the transcription, and then being resolved gradually to allow transcriptional bursting.

Gpn3 Is Essential for Cell Proliferation of Breast Cancer Cells Independent of Their Malignancy Degree.

Successful therapies for patients with breast cancer often lose their initial effectiveness. Thus, identifying new molecular targets is a constant goal in the fight against breast cancer. Gpn3 is a protein required for RNA polymerase II nuclear targeting in both yeast and human cells. We investigated here the effect of suppressing Gpn3 expression on cell proliferation in a progression series of isogenic cell lines derived from the nontumorigenic MCF-10A breast cells that recapitulate different stages of breast carcinogenesis. Gpn3 protein levels were comparable in all malignant derivatives of the nontumorigenic MCF-10A cells. shRNA-mediated inhibition of Gpn3 expression markedly decreased cell proliferation in all MCF-10A sublines. A fraction of the largest RNA polymerase II subunit Rpb1 was retained in the cytoplasm, but most Rpb1 remained nuclear after suppressing Gpn3 in all cell lines studied. Long-term proliferation experiments in cells with suppressed Gpn3 expression resulted in the eventual loss of all isogenic cell lines but MCF-10CA1d.cl1. In MCF-10CA1d.cl1 cells, Gpn3 knockdown reduced the proliferation of breast cancer stem cells as evaluated by mammosphere assays. After the identification that Gpn3 plays a key role in cell proliferation in mammary epithelial cells independent of the degree of transformation, we also analyzed the importance of Gpn3 in other human breast cancer cell lines from different subtypes. Gpn3 was also required for cell proliferation and nuclear translocation of RNA polymerase II in such cellular models. Altogether, our results show that Gpn3 is essential for breast cancer cell proliferation regardless of the transformation level, indicating that Gpn3 could be considered a molecular target for the development of new antiproliferative therapies. Importantly, our analysis of public data revealed that Gpn3 overexpression was associated with a significant decrease in overall survival in patients with estrogen receptor-positive and Human epidermal growth factor receptor 2 (HER2+) breast cancer, supporting our proposal that targeting Gpn3 could potentially benefit patients with breast cancer.

RNA-DNA and DNA-DNA base-pairing at the upstream edge of the transcription bubble regulate translocation of RNA polymerase and transcription rate.

Translocation of RNA polymerase (RNAP) along DNA may be rate-limiting for transcription elongation. The Brownian ratchet model posits that RNAP rapidly translocates back and forth until the post-translocated state is stabilized by NTP binding. An alternative model suggests that RNAP translocation is slow and poorly reversible. To distinguish between these two models, we take advantage of an observation that pyrophosphorolysis rates directly correlate with the abundance of the pre-translocated fraction. Pyrophosphorolysis by RNAP stabilized in the pre-translocated state by bacteriophage HK022 protein Nun was used as a reference point to determine the pre-translocated fraction in the absence of Nun. The stalled RNAP preferentially occupies the post-translocated state. The forward translocation rate depends, among other factors, on melting of the RNA–DNA base pair at the upstream edge of the transcription bubble. DNA–DNA base pairing immediately upstream from the RNA–DNA hybrid stabilizes the post-translocated state. This mechanism is conserved between E. coli RNAP and S. cerevisiae RNA polymerase II and is partially dependent on the lid domain of the catalytic subunit. Thus, the RNA–DNA hybrid and DNA reannealing at the upstream edge of the transcription bubble emerge as targets for regulation of the transcription elongation rate.

Quantifying the influence of 5′-RNA modifications on RNA polymerase I activity

For ensemble and single-molecule analyses of transcription, the use of synthetic transcription elongation complexes has been a versatile and powerful tool. However, structural analyses demonstrate that short RNA substrates, often employed in these assays, would occupy space within the RNA polymerase. Most commercial RNA oligonucleotides do not carry a 5′-triphosphate as would be present on a natural, de novo synthesized RNA. To examine the effects of 5′-moities on transcription kinetics, we measured nucleotide addition and 3′-dinucleotide cleavage by eukaryotic RNA polymerase I using 5′-hydroxyl and 5′-triphosphate RNA substrates. We found that 5′ modifications had no discernable effect on the kinetics of nucleotide addition; however, we observed clear, but modest, effects on the rate of backtracking and/or dinucleotide cleavage. These data suggest that the 5′-end may influence RNA polymerase translocation, consistent with previous prokaryotic studies, and these findings may have implications on kinetic barriers that confront RNA polymerases during the transition from initiation to elongation.

T7 RNA polymerase translocation is facilitated by a helix opening on the fingers domain that may also prevent backtracking.

Here, we studied the complete process of a viral T7 RNA polymerase (RNAP) translocation on DNA during transcription elongation by implementing extensive all-atom molecular dynamics (MD) simulations to construct a Markov state model (MSM). Our studies show that translocation proceeds in a Brownian motion, and the RNAP thermally transits among multiple metastable states. We observed non-synchronized backbone movements of the nucleic acid (NA) chains with the RNA translocation accomplished first, while the template DNA lagged. Notably, both the O-helix and Y-helix on the fingers domain play key roles in facilitating NA translocation through the helix opening. The helix opening allows a key residue Tyr639 to become inserted into the active site, which pushes the RNA–DNA hybrid forward. Another key residue, Phe644, coordinates the downstream template DNA motions by stacking and un-stacking with a transition nucleotide (TN) and its adjacent nucleotide. Moreover, the O-helix opening at pre-translocation (pre-trans) likely resists backtracking. To test this hypothesis, we computationally designed mutants of T7 RNAP by replacing the amino acids on the O-helix with counterpart residues from a mitochondrial RNAP that is capable of backtracking. The current experimental results support the hypothesis.

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.

Regulation of RNA Polymerase Translocation by the RNA and DNA Hybridization at the Upstream Edge of the Transcription Bubble

Regulation of RNA Polymerase Translocation by the RNA and DNA Hybridization at the Upstream Edge of the Transcription Bubble

Structural basis of transcription arrest by coliphage HK022 Nun in an Escherichia coli RNA polymerase elongation complex.

Coliphage HK022 Nun blocks superinfection by coliphage λ by stalling RNA polymerase (RNAP) translocation specifically on λ DNA. To provide a structural framework to understand how Nun blocks RNAP translocation, we determined structures of Escherichia coli RNAP ternary elongation complexes (TECs) with and without Nun by single-particle cryo-electron microscopy. Nun fits tightly into the TEC by taking advantage of gaps between the RNAP and the nucleic acids. The C-terminal segment of Nun interacts with the RNAP β and β' subunits inside the RNAP active site cleft as well as with nearly every element of the nucleic acid scaffold, essentially crosslinking the RNAP and the nucleic acids to prevent translocation, a mechanism supported by the effects of Nun amino acid substitutions. The nature of Nun interactions inside the RNAP active site cleft suggests that RNAP clamp opening is required for Nun to establish its interactions, explaining why Nun acts on paused TECs.

Optical tweezers studies of transcription by eukaryotic RNA polymerases.

Transcription is the first step in the expression of genetic information and it is carried out by large macromolecular enzymes called RNA polymerases. Transcription has been studied for many years and with a myriad of experimental techniques, ranging from bulk studies to high-resolution transcript sequencing. In this review, we emphasise the advantages of using single-molecule techniques, particularly optical tweezers, to study transcription dynamics. We give an overview of the latest results in the single-molecule transcription field, focusing on transcription by eukaryotic RNA polymerases. Finally, we evaluate recent quantitative models that describe the biophysics of RNA polymerase translocation and backtracking dynamics.

RNA Polymerase Translocation in Processive Transcription Elongation and Pausing: Dynamics, Force-Dependence, and Modulation by Sequence-Specific RNAP-DNA Interactions

During transcription elongation, E. coli RNA polymerase (RNAP) alternates between periods of processive nucleotide addition and periods of pausing. Various mechanisms promote pausing. In particular, increased pausing at a “consensus pause element” of the form G-10Y-1G+1 (where Y-1 corresponds to the 3′ RNA end) has been described (Science 344:1042, 2014; Science 344:1285, 2014). Such pause sites have been shown to exhibit increased efficiency in the RNAP βD446A mutant; this effect can be understood from the interaction between WT RNAP βD446 and G+1, which stabilizes RNAP in a post-translocated register in the presence of a G+1, and thus decreases pausing (Science 344:1285, 2014).In this work, we use high-resolution optical tweezers to characterize the dynamics of both processive nucleotide elongation and pausing by WT and βD446A RNAP at very high spatial and temporal resolutions, under various force conditions. Our ability to resolve sub-second pausing events provide new insights into the dynamics of RNAP translocation and pausing during elongation, and their modulation by sequence-specific RNAP-DNA interactions.

Probing the structure of Nun transcription arrest factor bound to RNA polymerase

The coliphage HK022 protein Nun transcription elongation arrest factor inhibits RNA polymerase translocation. In vivo, Nun acts specifically to block transcription of the coliphage λ chromosome. Using in vitro assays, we demonstrate that Nun cross-links RNA in an RNA:DNA hybrid within a ternary elongation complex (TEC). Both the 5' and the 3' ends of the RNA cross-link Nun, implying that Nun contacts RNA polymerase both at the upstream edge of the RNA:DNA hybrid and in the vicinity of the catalytic center. This finding suggests that Nun may inhibit translocation by more than one mechanism. Transcription elongation factor GreA efficiently blocked Nun cross-linking to the 3' end of the transcript, whereas the highly homologous GreB factor did not. Surprisingly, both factors strongly suppressed Nun cross-linking to the 5' end of the RNA, suggesting that GreA and GreB can enter the RNA exit channel as well as the secondary channel, where they are known to bind. These findings extend the known action mechanism for these ubiquitous cellular factors.

Structural basis of viral RNA-dependent RNA polymerase catalysis and translocation

Viral RNA-dependent RNA polymerases (RdRPs) play essential roles in viral genome replication and transcription. We previously reported several structural states of the poliovirus RdRP nucleotide addition cycle (NAC) that revealed a unique palm domain-based active site closure mechanism and proposed a six-state NAC model including a hypothetical state representing translocation intermediates. Using the RdRP from another human enterovirus, enterovirus 71, here we report seven RdRP elongation complex structures derived from a crystal lattice that allows three NAC events. These structures suggested a key order of events in initial NTP binding and NTP-induced active site closure and revealed a bona fide translocation intermediate featuring asymmetric movement of the template-product duplex. Our work provides essential missing links in understanding NTP recognition and translocation mechanisms in viral RdRPs and emphasizes the uniqueness of the viral RdRPs compared with other processive polymerases.