Release Of RNA Polymerase Research Articles

Structural basis of σ54 displacement and promoter escape in bacterial transcription.

Gene transcription is a fundamental cellular process carried out by RNA polymerase (RNAP). Transcription initiation is highly regulated, and in bacteria, transcription initiation is mediated by sigma (σ) factors. σ recruits RNAP to the promoter DNA region, located upstream of the transcription start site (TSS) and facilitates open complex formation, where double-stranded DNA is opened up into a transcription bubble and template strand DNA is positioned inside RNAP for initial RNA synthesis. During initial transcription, RNAP remains bound to σ and upstream DNA, presumably with an enlarging transcription bubble. The release of RNAP from upstream DNA is required for promoter escape and processive transcription elongation. Bacteria sigma factors can be broadly separated into two classes with the majority belonging to the σ70 class, represented by the σ70 that regulates housekeeping genes. σ54 forms a class on its own and regulates stress response genes. Extensive studies on σ70 have revealed the molecular mechanisms of the σ70 dependent process while how σ54 transitions from initial transcription to elongation is currently unknown. Here, we present a series of cryo-electron microscopy structures of the RNAP-σ54 initial transcribing complexes with progressively longer RNA, which reveal structural changes that lead to promoter escape. Our data show that initially, the transcription bubble enlarges, DNA strands scrunch, reducing the interactions between σ54 and DNA strands in the transcription bubble. RNA extension and further DNA scrunching help to release RNAP from σ54 and upstream DNA, enabling the transition to elongation.

Ligand Modulates Cross-Coupling between Riboswitch Folding and Transcriptional Pausing

Numerous classes of riboswitches have been found to regulate bacterial gene expression in response to physiological cues, offering new paths to antibacterial drugs. As common studies of isolated riboswitches lack the functional context of the transcription machinery, we here combine single-molecule, biochemical, and simulation approaches to investigate the coupling between co-transcriptional folding of the pseudoknot-structured preQ1 riboswitch and RNA polymerase (RNAP) pausing. We show that pausing at a site immediately downstream of the riboswitch requires a ligand-free pseudoknot in the nascent RNA, a precisely spaced sequence resembling the pause consensus, and electrostatic and steric interactions with the RNAP exit channel. While interactions with RNAP stabilize the native fold of the riboswitch, binding of the ligand signals RNAP release from the pause. Our results demonstrate that the nascent riboswitch and its ligand actively modulate the function of RNAP and vice versa, a paradigm likely to apply to other cellular RNA transcripts.

Regulation of the Alternative Splicing and Function of Cyclin T1 by the Serine‐Arginine‐Rich Protein ASF/SF2

Positive transcription elongation factor-b (P-TEFb) is required for the release of RNA polymerase II (RNAPII) from its pause near the gene promoters and thus for efficient proceeding to the transcription elongation. It consists of two core subunits-CDK9 and one of T-typed or K-typed cyclin, of which, cyclin T1/CDK9 is the major and most studied combination. We have previously identified a novel splice variant of cyclin T1, cyclin T1b, which negatively regulates the transcription elongation of HIV-1 genes as well as several host genes. In this study, we revealed the serine-arginine-rich protein, ASF/SF2, as a regulatory factor of the alternative splicing of cyclin T1 gene. ASF/SF2 promotes the production of cyclin T1b versus cyclin T1a and regulates the expression of cyclin T1-depedent genes at the transcription level. We further found that a cis-element on exon 8 is responsible for the skipping of exon 7 mediated by ASF/SF2. Collectively, ASF/SF2 is identified as a splicing regulator of cyclin T1, which contributes to the control of the subsequent transcription events. J. Cell. Biochem. 118: 4020-4032, 2017. © 2017 Wiley Periodicals, Inc.

Bacillus subtilis 6S-2 RNA serves as a template for short transcripts in vivo

The global transcriptional regulator 6S RNA is abundant in a broad range of bacteria. The RNA competes with DNA promoters for binding to the housekeeping RNA polymerase (RNAP) holoenzyme. When bound to RNAP, 6S RNA serves as a transcription template for RNAP in an RNA-dependent RNA polymerization reaction. The resulting short RNA transcripts (so-called product RNAs = pRNAs) can induce a stable structural rearrangement of 6S RNA when reaching a certain length. This rearrangement leads to the release of RNAP and thus the recovery of transcription at DNA promoters. While most bacteria express a single 6S RNA, some harbor a second 6S RNA homolog (termed 6S-2 RNA in Bacillus subtilis). Bacillus subtilis 6S-2 RNA was recently shown to exhibit essentially all hallmark features of a bona fide 6S RNA in vitro, but evidence for the synthesis of 6S-2 RNA-derived pRNAs in vivo has been lacking so far. This raised the question of whether the block of RNAP by 6S-2 RNA might be lifted by a mechanism other than pRNA synthesis. However, here we demonstrate that 6S-2 RNA is able to serve as a template for pRNA synthesis in vivo. We verify this finding by using three independent approaches including a novel primer extension assay. Thus, we demonstrate the first example of an organism that expresses two distinct 6S RNAs that both exhibit all mechanistic features defined for this type of regulatory RNA.

Structural and mechanistic characterization of 6S RNA from the hyperthermophilic bacterium Aquifex aeolicus.

Bacterial 6S RNAs competitively inhibit binding of RNA polymerase (RNAP) holoenzymes to DNA promoters, thereby globally regulating transcription. RNAP uses 6S RNA itself as a template to synthesize short transcripts, termed pRNAs (product RNAs). Longer pRNAs (approx.≥10nt) rearrange the 6S RNA structure and thereby disrupt the 6S RNA:RNAP complex, which enables the enzyme to resume transcription at DNA promoters. We studied 6S RNA of the hyperthermophilic bacterium Aquifex aeolicus, representing the thermodynamically most stable 6S RNA known so far. Applying structure probing and NMR, we show that the RNA adopts the canonical rod-shaped 6S RNA architecture with little structure formation in the central bulge (CB) even at moderate temperatures (≤37°C). 6S RNA:pRNA complex formation triggers an internal structure rearrangement of 6S RNA, i.e. formation of a so-called central bulge collapse (CBC) helix. The persistence of several characteristic NMR imino proton resonances upon pRNA annealing demonstrates that defined helical segments on both sides of the CB are retained in the pRNA-bound state, thus representing a basic framework of the RNA's architecture. RNA-seq analyses revealed pRNA synthesis from 6S RNA in A.aeolicus, identifying 9 to ∼17-mers as the major length species. A.aeolicus 6S RNA can also serve as a template for invitro pRNA synthesis by RNAP from the mesophile Bacillus subtilis. Binding of a synthetic pRNA to A.aeolicus 6S RNA blocks formation of 6S RNA:RNAP complexes. Our findings indicate that A.aeolicus 6S RNA function in its hyperthermophilic host is mechanistically identical to that of other bacterial 6S RNAs. The use of artificial pRNA variants, designed to disrupt helix P2 from the 3'-CB instead of the 5'-CB but preventing formation of the CBC helix, indicated that the mechanism of pRNA-induced RNAP release has been evolutionarily optimized for transcriptional pRNA initiation in the 5'-CB.

Nucleotide excision repair (NER) machinery recruitment by the transcription-repair coupling factor involves unmasking of a conserved intramolecular interface

Transcription-coupled DNA repair targets DNA lesions that block progression of elongating RNA polymerases. In bacteria, the transcription-repair coupling factor (TRCF; also known as Mfd) SF2 ATPase recognizes RNA polymerase stalled at a site of DNA damage, removes the enzyme from the DNA, and recruits the Uvr(A)BC nucleotide excision repair machinery via UvrA binding. Previous studies of TRCF revealed a molecular architecture incompatible with UvrA binding, leaving its recruitment mechanism unclear. Here, we examine the UvrA recognition determinants of TRCF using X-ray crystallography of a core TRCF-UvrA complex and probe the conformational flexibility of TRCF in the absence and presence of nucleotides using small-angle X-ray scattering. We demonstrate that the C-terminal domain of TRCF is inhibitory for UvrA binding, but not RNA polymerase release, and show that nucleotide binding induces concerted multidomain motions. Our studies suggest that autoinhibition of UvrA binding in TRCF may be relieved only upon engaging the DNA damage.

Chromatin association and regulation of rDNA transcription by the Ras-family protein RasL11a

RasL11a and RasL11b are Ras super-family proteins of unknown function. Here, we show that RasL11a is a chromatin-associated modulator of pre-ribosomal RNA (pre-rRNA) synthesis. RasL11a was found in the nucleolus of interphase mouse fibroblasts, where it co-localized with the RNA polymerase I-specific transcription factor UBF. Similar to UBF, RasL11a also marked the active subset of rDNA repeats (also called nucleolar organizers, or NORs) on mitotic chromosomes. In cells, RasL11a existed in stable complexes with UBF and, as shown by chromatin immunoprecipitation, distributed along the rDNA transcription unit. Upon treatment of cells with actinomycin D, RasL11a and UBF persisted on the transcription unit beyond the release of RNA polymerase I, and remained co-localized in peri-nucleolar cap structures. Ectopic expression of RasL11a enhanced pre-rRNA levels in cells, whereas RasL11a knockdown had the opposite effect. In transient transfection experiments, RasL11a enhanced the transcriptional activity of an RNA polymerase I-specific reporter controlled by the rDNA enhancer/promoter region. We speculate that RasL11a acts in concert with UBF to facilitate initiation and/or elongation by RNA polymerase I in response to specific upstream stimuli.

Model of gene transcription including the return of a RNA polymerase to the beginning of a transcriptional cycle

The gene transcription occurs via the RNA polymerase (RNAP) recruitment on the DNA promoter sequence, formation of a locally open DNA chain, promoter escape, steps of the RNA synthesis, and RNA and RNAP release after reading the final DNA base. Just after the end of the RNA synthesis, RNAP surrounds the closed DNA chain and may diffuse along DNA, desorb, or reach the promoter and start the RNA-synthesis cycle again. We present a generic kinetic model taking the latter steps into account and show analytically and by Monte Carlo simulations that it predicts transcriptional bursts even in the absence of explicit regulation of the transcription by master proteins.

A genomic analysis of RNA polymerase II modification and chromatin architecture related to 3′ end RNA polyadenylation

Genomic analyses have been applied extensively to analyze the process of transcription initiation in mammalian cells, but less to transcript 3' end formation and transcription termination. We used a novel approach to prepare 3' end fragments from polyadenylated RNA, and mapped the position of the poly(A) addition site using oligonucleotide arrays tiling 1% of the human genome. This approach revealed more 3' ends than had been annotated. The distribution of these ends relative to RNA polymerase II (PolII) and di- and trimethylated lysine 4 and lysine 36 of histone H3 was compared. A substantial fraction of unannotated 3' ends of RNA are intronic and antisense to the embedding gene. Poly(A) ends of annotated messages lie on average 2 kb upstream of the end of PolII binding (termination). Near the termination sites, and in some internal sites, unphosphorylated and C-terminal domain (CTD) serine 2 phosphorylated PolII (POLR2A) accumulate, suggesting pausing of the polymerase and perhaps dephosphorylation prior to release. Lysine 36 trimethylation occurs across transcribed genes, sometimes alternating with stretches of DNA in which lysine 36 dimethylation is more prominent. Lysine 36 methylation decreases at or near the site of polyadenylation, sometimes disappearing before disappearance of phosphorylated RNA PolII or release of PolII from DNA. Our results suggest that transcription termination loss of histone 3 lysine 36 methylation and later release of RNA polymerase. The latter is often associated with polymerase pausing. Overall, our study reveals extensive sites of poly(A) addition and provides insights into the events that occur during 3' end formation.

Chromatin-mediated regulation of nucleolar structure and RNA Pol I localization by TOR.

The target of rapamycin (TOR) protein is a conserved regulator of ribosome biogenesis, an important process for cell growth and proliferation. However, how TOR is involved remains poorly understood. In this study, we find that rapamycin and nutrient starvation, conditions inhibiting TOR, lead to significant nucleolar size reduction in both yeast and mammalian cells. In yeast, this morphological change is accompanied by release of RNA polymerase I (Pol I) from the nucleolus and inhibition of ribosomal DNA (rDNA) transcription. We also present evidence that TOR regulates association of Rpd3-Sin3 histone deacetylase (HDAC) with rDNA chromatin, leading to site-specific deacetylation of histone H4. Moreover, histone H4 hypoacetylation mutations cause nucleolar size reduction and Pol I delocalization, while rpd3Delta and histone H4 hyperacetylation mutations block the nucleolar changes as a result of TOR inhibition. Taken together, our results suggest a chromatin-mediated mechanism by which TOR modulates nucleolar structure, RNA Pol I localization and rRNA gene expression in response to nutrient availability.

Template Strand Switching by T7 RNA Polymerase

T7 RNA polymerase (RNAP) is able to traverse a variety of discontinuities in the template (T) strand of duplex DNA, including nicks, gaps, and branched junctions in which the 3' end of the T strand is not complementary to the non-template (NT) strand. The products represent a faithful copy of the T strand, with no insertions or deletions. On double-stranded templates having protruding 3' ends the polymerase is able to insert the free 3' end of the NT strand and to utilize this as a new T strand ("turn around transcription"), resulting in the anomalous production of high molecular weight transcripts. The capacity of T7 RNAP to bypass interruptions in the T strand depends upon the stability of the elongation complex. Sequences that are expected to stabilize a local RNA:DNA hybrid (such as the presence of a C6 tract in the T strand) dramatically reduce dissociation of the RNAP while still allowing the enzyme to insert a new 3' end. Similar effects on RNAP release are observed when the enzyme reaches the end of a template (i.e. when synthesizing runoff products), resulting in markedly different yields of RNA product during multiple rounds of transcription.

Reversible Phosphorylation of the C-terminal Domain of RNA Polymerase II

Reversible Phosphorylation of the C-terminal Domain of RNA Polymerase II

Release of RNA polymerase from vero cell mitochondria after herpes simplex virus type 1 infection

Infection of Vero cells with herpes simplex virus type 1 results in the appearance in soluble extracts of a DNA primase activity. The partially purified enzyme, Mr, approximately 100,000, is identical in resistance to alpha-amanitin, pH profile, Mg2+ dependence, salt sensitivity, and KmATP to the catalytic core of Vero cell mitochondrial RNA polymerase. Moreover, the products synthesized are those expected of an RNA polymerase rather than a DNA primase. Inasmuch as the enzyme is not present in soluble extracts of uninfected Vero cells, we presume that the specific appearance of RNA polymerase in extracts of herpesvirus-infected cells results from infection-induced disruption of the mitochondrial membrane, followed by release of the enzyme into the cytosol.

Transcription termination in Escherichia coli: Measurement of the rate of enzyme release from rho-independent terminators

The termination/release phase of transcription must involve at least three major steps: cessation of elongation; release of the transcript; and release of the RNA polymerase. We have devised a novel method for measuring the rate of Escherichia coli RNA polymerase release during transcription termination. The method is based on a kinetic analysis of the rate of RNA synthesis during steady-state transcription. Using this method with defined transcription units, we have found that RNA polymerase release occurs rapidly from several rho-independent terminators. Enzyme release from the T7 early terminator occurs within 13(± 3) seconds of the cessation of elongation. Neither nusA protein nor supercoiling of the DNA template affects the rate of enzyme release. However, addition of excess sigma factor significantly increases the rate of enzyme recycling during the steady state. Since added sigma factor does not alter the rates of initiation and elongation by E. coli RNA polymerase holoenzyme, it appears that sigma factor stimulates one or more steps in the termination/release process and reduces the rate of enzyme release to a few seconds. We present evidence that suggests sigma may be directly involved in catalyzing release of the core RNA polymerase from the DNA template during transcription termination. The rapid rates of enzyme release we measure make it difficult to be certain of the exact pathway of events that occur in the termination/release phase of transcription. The most plausible pathway involves initial release of the RNA transcript followed by release of core RNA polymerase from the DNA. Studies on the properties of core polymerase-RNA complexes indicate that core polymerase and the RNA transcript probably do not dissociate as a complex from the terminator. Furthermore, these core-RNA complexes are too stable to represent significant intermediates in the termination/release pathway, at least in the early steps of the reaction.

In vitro transcription of E. coli tRNA genes

Transcription of tRNA genes carried by transducing bacteriophages phi80psu3+ (tRNA1Tyr) and lambdah80T (tRNA2Tyr, tRNA2Glysu36+, tRNA3Thr) was studied in vitro in a system consisting of whole bacteriophage DNA and purified RNA polymerase. In contrast to unusual requirements for tRNA1Tyr gene transcription from DNA fragments, the transcription on whole bacteriophage DNA was found to be relatively not salt sensitive, did not require glycerol and rifampicin-resistant complexes with RNA polymerase were formed in the absence of nucleoside triphosphates. Termination factor rho stimulated the transcription of the tRNA genes as well as that of 4S RNA on lambdah80T DNA template. The stimulatory effect of rho was abolished by rifampicin and seems to be due to the release of RNA polymerase and reinitiation of transcription.

The effect of modification of T7 DNA by the carcinogen N-1-acetylaminofluorene: termination of transcription in vitro.

To study the effects of N-2-acetylaminofluorene (AAF) modification of DNA on transcription, purified DNA from bacteriophage T7 was modified in vitro to varying extent with AAF and transcribed by DNA-dependent RNA polymerase from Escherichia coli. The main effects of AAF modification on transcription are a marked inhibition of the rate and extent of trna synthesis with relatively little effect on initiation except at very high AAF doses. Calibration of the percent modification with [14-C]AAF and analysis of the size of the RNA product by double isotope labeling and polyacrylamide gel electrophoresis support the following mechanism of transcription inhibition: most of the AAF residues bound to the coding strand of the DNA cause premature termination of transcription, at or near the site of modification, with release of RNA polymerase. This results in the production of shorter RNA chains with increasing amounts of bound carcinogen. The data are consistent with there being no reinitiation and/or synthesis of RNA distal to the AAF-modification site.

New evidence that release of RNA polymerase in isolated nuclei requires transcription

Summary RNA polymerases solubilized by transcription in isolated nuclei at various (NH 4 ) 2 SO 4 concentrations were analyzed to determine the proportion of amanitin-sensitive and amanitin-resistant activity. Ionic conditions that stimulated amanitin-sensitive transcription in isolated nuclei increased the amount of RNA polymerase II released. Approximately equal amounts of RNA polymerase I were released at all ionic strengths tested.