scholarly journals Structural visualization of de novo initiation of RNA polymerase II transcription

2021 ◽  
Author(s):  
Chun Yang ◽  
Rina Fujiwara ◽  
Hee Jong Kim ◽  
Jose J Gorbea Col&oacuten ◽  
Stefan Steimle ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
De Novo ◽  
Base Pairs ◽  
Pol Ii ◽  
Promoter Escape ◽  
General Transcription Factor ◽  
Short Transcript ◽  
Transition To Elongation ◽  
Rna Polymerase Ii Transcription

Structural studies of the initiation-elongation transition of RNA polymerase II (pol II) transcription were previously facilitated by the use of synthetic oligonucleotides. Here we report structures of initiation complexes de novo converted from pre-initiation complex (PIC) through catalytic activities and stalled at different template positions. Contrary to previous models, the closed-to-open promoter transition was accompanied by a large positional change of the general transcription factor TFIIH which became in closer proximity to TFIIE for the active delivery of the downstream DNA to the pol II active center. The initially-transcribing complex (ITC) reeled over 80 base pairs of the downstream DNA by scrunching, while retaining the fixed upstream contact, and underwent the transition to elongation when it encountered promoter-proximal pol II from a preceding round of transcription. TFIIH is therefore conducive to promoter melting, TSS scanning, and promoter escape, extending far beyond synthesis of a short transcript.

Structural Biology of RNA Polymerase II Transcription: 20 Years On

2020 ◽  
Vol 36 (1) ◽  
pp. 1-34 ◽  
Author(s):  
Sara Osman ◽  
Patrick Cramer
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Transcription Initiation ◽  
Focal Point ◽  
Dna Lesions ◽  
Cellular Regulation ◽  
Pol Ii ◽  
Associated Proteins ◽  
Rna Polymerase Ii Transcription

Gene transcription by RNA polymerase II (Pol II) is the first step in the expression of the eukaryotic genome and a focal point for cellular regulation during development, differentiation, and responses to the environment. Two decades after the determination of the structure of Pol II, the mechanisms of transcription have been elucidated with studies of Pol II complexes with nucleic acids and associated proteins. Here we provide an overview of the nearly 200 available Pol II complex structures and summarize how these structures have elucidated promoter-dependent transcription initiation, promoter-proximal pausing and release of Pol II into active elongation, and the mechanisms that Pol II uses to navigate obstacles such as nucleosomes and DNA lesions. We predict that future studies will focus on how Pol II transcription is interconnected with chromatin transitions, RNA processing, and DNA repair.

scholarly journals Live-cell single particle imaging reveals the role of RNA polymerase II in histone H2A.Z eviction

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Anand Ranjan ◽  
Vu Q Nguyen ◽  
Sheng Liu ◽  
Jan Wisniewski ◽  
Jee Min Kim ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Single Molecule ◽  
Transcription Initiation ◽  
General Mechanism ◽  
Pol Ii ◽  
Promoter Escape ◽  
Genome Wide ◽  
A Genome ◽  
Particle Imaging

The H2A.Z histone variant, a genome-wide hallmark of permissive chromatin, is enriched near transcription start sites in all eukaryotes. H2A.Z is deposited by the SWR1 chromatin remodeler and evicted by unclear mechanisms. We tracked H2A.Z in living yeast at single-molecule resolution, and found that H2A.Z eviction is dependent on RNA Polymerase II (Pol II) and the Kin28/Cdk7 kinase, which phosphorylates Serine 5 of heptapeptide repeats on the carboxy-terminal domain of the largest Pol II subunit Rpb1. These findings link H2A.Z eviction to transcription initiation, promoter escape and early elongation activities of Pol II. Because passage of Pol II through +1 nucleosomes genome-wide would obligate H2A.Z turnover, we propose that global transcription at yeast promoters is responsible for eviction of H2A.Z. Such usage of yeast Pol II suggests a general mechanism coupling eukaryotic transcription to erasure of the H2A.Z epigenetic signal.

scholarly journals RNA Polymerase II (Pol II)-TFIIF and Pol II-Mediator Complexes: the Major Stable Pol II Complexes and Their Activity in Transcription Initiation and Reinitiation

2004 ◽  
Vol 24 (4) ◽  
pp. 1709-1720 ◽  
Author(s):  
P. Geetha Rani ◽  
Jeffrey A. Ranish ◽  
Steven Hahn
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Transcription Initiation ◽  
Specific Activity ◽  
Pol Ii ◽  
Transcription Activity ◽  
General Transcription Factor ◽  
Nuclear Extracts ◽  
Transcription Reinitiation ◽  
Stable Forms

ABSTRACT Protein purification and depletion studies were used to determine the major stable forms of RNA polymerase II (Pol II) complexes found in Saccharomyces cerevisiae nuclear extracts. About 50% of Pol II is found associated with the general transcription factor TFIIF (Pol II-TFIIF), and about 20% of Pol II is associated with Mediator (Pol-Med). No Pol II-Med-TFIIF complex was observed. The activity of Pol II and the purified Pol II complexes in transcription initiation and reinitiation was investigated by supplementing extracts depleted of either total Pol II or total TFIIF with purified Pol II or the Pol II complexes. We found that all three forms of Pol II can complement Pol II-depleted extracts for transcription initiation, but Pol II-TFIIF has the highest specific activity. Similarly, Pol II-TFIIF has a much higher specific activity than TFIIF for complementation of TFIIF transcription activity. Although the Pol II-TFIIF and Pol II-Med complexes were stable when purified, we found these complexes were dynamic in extracts under transcription conditions, with a single polymerase capable of exchanging bound Mediator and TFIIF. Using a purified system to examine transcription reinitiation, we found that Pol II-TFIIF was active in promoting multiple rounds of transcription while Pol II-Med was nearly inactive. These results suggest that both the Pol II-Med and Pol II-TFIIF complexes can be recruited for transcription initiation but that only the Pol II-TFIIF complex is competent for transcription reinitiation.

scholarly journals Studies of Nematode TFIIE Function Reveal a Link between Ser-5 Phosphorylation of RNA Polymerase II and the Transition from Transcription Initiation to Elongation

2001 ◽  
Vol 21 (1) ◽  
pp. 1-15 ◽  
Author(s):  
Seiji Yamamoto ◽  
Yoshinori Watanabe ◽  
Peter J. van der Spek ◽  
Tomomichi Watanabe ◽  
Hiroyuki Fujimoto ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Transcription Initiation ◽  
In Vitro Transcription ◽  
Pol Ii ◽  
Transcription System ◽  
Carboxy Terminal ◽  
Transition To Elongation ◽  
Ctd Phosphorylation

ABSTRACT The general transcription factor TFIIE plays important roles in transcription initiation and in the transition to elongation. However, little is known about its function during these steps. Here we demonstrate for the first time that TFIIH-mediated phosphorylation of RNA polymerase II (Pol II) is essential for the transition to elongation. This phosphorylation occurs at serine position 5 (Ser-5) of the carboxy-terminal domain (CTD) heptapeptide sequence of the largest subunit of Pol II. In a human in vitro transcription system with a supercoiled template, this process was studied using a human TFIIE (hTFIIE) homolog from Caenorhabditis elegans (ceTFIIEα and ceTFIIEβ). ceTFIIEβ could partially replace hTFIIEβ, whereas ceTFIIEα could not replace hTFIIEα. We present the studies of TFIIE binding to general transcription factors and the effects of subunit substitution on CTD phosphorylation. As a result, ceTFIIEα did not bind tightly to hTFIIEβ, and ceTFIIEβ showed a similar profile for binding to its human counterpart and supported an intermediate level of CTD phosphorylation. Using antibodies against phosphorylated serine at either Ser-2 or Ser-5 of the CTD, we found that ceTFIIEβ induced Ser-5 phosphorylation very little but induced Ser-2 phosphorylation normally, in contrast to wild-type hTFIIE, which induced phosphorylation at both Ser-2 and Ser-5. In transcription transition assays using a linear template, ceTFIIEβ was markedly defective in its ability to support the transition to elongation. These observations provide evidence of TFIIE involvement in the transition and suggest that Ser-5 phosphorylation is essential for Pol II to be in the processive elongation form.

scholarly journals RNA Polymerase II-Controlled Expression of Antigenomic RNA Enhances the Rescue Efficacies of Two Different Members of the Mononegavirales Independently of the Site of Viral Genome Replication

2006 ◽  
Vol 80 (12) ◽  
pp. 5708-5715 ◽  
Author(s):  
Arnold Martin ◽  
Peter Staeheli ◽  
Urs Schneider
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Measles Virus ◽  
De Novo ◽  
Hammerhead Ribozyme ◽  
Genome Replication ◽  
Pol Ii ◽  
Pol I ◽  
Replication Phase ◽  
Viral Genome Replication

ABSTRACT De novo generation of negative-strand RNA viruses depends on the efficient expression of antigenomic RNA (cRNA) from cDNA. To improve the rescue system of Borna disease virus (BDV), a member of the Mononegavirales with a nuclear replication phase, we evaluated different RNA polymerase (Pol) promoters for viral cRNA expression. Human and mouse Pol I promoters did not increase the recovery rate of infectious BDV from cDNA compared to the originally employed T7 RNA polymerase system. In contrast, expression of viral cRNA under the control of an RNA Pol II promoter increased the rescue efficacy by nearly 20-fold. Similarly, rescue of measles virus (MV), a member of the Mononegavirales with a cytoplasmic replication phase, was strongly improved by Pol II-controlled expression of viral cRNA. Analysis of transcription levels derived from different promoters suggested that the rescue-enhancing function of the Pol II promoter was due mainly to enhanced cRNA synthesis from the plasmid. Remarkably, correct 5′-terminal processing of Pol II-transcribed cRNA by a hammerhead ribozyme was not necessary for efficient rescue of BDV or MV. The correct 5′ termini were reconstituted during replication of the artificially prolonged cRNA, indicating that the BDV and MV replicase complexes are able to recognize internal viral replication signals.

scholarly journals CDK12 globally stimulates RNA polymerase II transcription elongation and carboxyl-terminal domain phosphorylation

2020 ◽  
Vol 48 (14) ◽  
pp. 7712-7727
Author(s):  
Michael Tellier ◽  
Justyna Zaborowska ◽  
Livia Caizzi ◽  
Eusra Mohammad ◽  
Taras Velychko ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Transcription Elongation ◽  
Pol Ii ◽  
Terminal Domain ◽  
Carboxyl Terminal Domain ◽  
Genome Wide ◽  
A Genome ◽  
Carboxyl Terminal ◽  
Rna Polymerase Ii Transcription

Abstract Cyclin-dependent kinase 12 (CDK12) phosphorylates the carboxyl-terminal domain (CTD) of RNA polymerase II (pol II) but its roles in transcription beyond the expression of DNA damage response genes remain unclear. Here, we have used TT-seq and mNET-seq to monitor the direct effects of rapid CDK12 inhibition on transcription activity and CTD phosphorylation in human cells. CDK12 inhibition causes a genome-wide defect in transcription elongation and a global reduction of CTD Ser2 and Ser5 phosphorylation. The elongation defect is explained by the loss of the elongation factors LEO1 and CDC73, part of PAF1 complex, and SPT6 from the newly-elongating pol II. Our results indicate that CDK12 is a general activator of pol II transcription elongation and indicate that it targets both Ser2 and Ser5 residues of the pol II CTD.

scholarly journals Evidence Supporting That RNA Polymerase II Catalyzes De Novo Transcription Using Potato Spindle Tuber Viroid Circular RNA Templates

Viruses ◽  
2020 ◽  
Vol 12 (4) ◽  
pp. 371
Author(s):  
Shachinthaka D. Dissanayaka Mudiyanselage ◽  
Ying Wang
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
De Novo ◽  
Unit Length ◽  
Potato Spindle Tuber Viroid ◽  
Circular Rna ◽  
Asymmetric Rolling ◽  
Pol Ii ◽  
Rolling Circle ◽  
Dna And Rna

Transcription is a fundamental process that mediates the interplay between genetic information and phenotype. Emerging evidence indicates that RNA polymerase II (Pol II) can catalyze transcription using both DNA and RNA templates. It is well established that Pol II initiates de novo transcription on DNA templates. However, it is unclear whether Pol II performs de novo transcription or relies on primers for initiation (primed transcription) on RNA templates. Using potato spindle tuber viroid (PSTVd) as a model, we presented evidence showing that circular PSTVd templates are critical for the synthesis of longer-than-unit-length (−)-strand products, which supports the de novo transcription based on the asymmetric rolling circle model of PSTVd replication. We further showed that the crucial factor for primed transcription, transcription factor IIS (TFIIS), is dispensable for PSTVd replication in cells. Together, our data support the de novo transcription on PSTVd RNA templates catalyzed by Pol II. This result has significant implications in understanding the mechanism and machinery underlying Pol II-catalyzed transcription using other RNA templates.

scholarly journals A global change in RNA polymerase II pausing during the Drosophila midblastula transition

eLife ◽  
2013 ◽  
Vol 2 ◽  
Author(s):  
Kai Chen ◽  
Jeff Johnston ◽  
Wanqing Shao ◽  
Samuel Meier ◽  
Cynthia Staber ◽  
...  
Keyword(s):  
Global Change ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
De Novo ◽  
Dynamic Properties ◽  
Core Promoter ◽  
Midblastula Transition ◽  
Pol Ii ◽  
Zygotic Transcription ◽  
Pol Ii Pausing

Massive zygotic transcription begins in many organisms during the midblastula transition when the cell cycle of the dividing egg slows down. A few genes are transcribed before this stage but how this differential activation is accomplished is still an open question. We have performed ChIP-seq experiments on tightly staged Drosophila embryos and show that massive recruitment of RNA polymerase II (Pol II) with widespread pausing occurs de novo during the midblastula transition. However, ∼100 genes are strongly occupied by Pol II before this timepoint and most of them do not show Pol II pausing, consistent with a requirement for rapid transcription during the fast nuclear cycles. This global change in Pol II pausing correlates with distinct core promoter elements and associates a TATA-enriched promoter with the rapid early transcription. This suggests that promoters are differentially used during the zygotic genome activation, presumably because they have distinct dynamic properties.

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