scholarly journals Cyclin T1/CDK9 Interacts with Influenza A Virus Polymerase and Facilitates Its Association with Cellular RNA Polymerase II

2010 ◽  
Vol 84 (24) ◽  
pp. 12619-12627 ◽  
Author(s):  
Junjie Zhang ◽  
Gang Li ◽  
Xin Ye
Keyword(s):  
Influenza Virus ◽  
Rna Polymerase ◽  
Viral Replication ◽  
Rna Polymerase Ii ◽  
Transcription Initiation ◽  
Influenza A ◽  
Viral Transcription ◽  
Rna Dependent Rna Polymerase ◽  
Pol Ii ◽  
Cyclin T1

ABSTRACT Influenza virus RNA-dependent RNA polymerase scavenges the 5′ cap from host pre-mRNA to prime viral transcription initiation. It is also well established that viral RNA-dependent RNA polymerase (vRNP) associates with cellular RNA polymerase II (Pol II), on which viral replication depends. Here we report that cyclin T1/CDK9 can interact with influenza virus polymerase and facilitate its association with cellular Pol II. The immunodepletion of cyclin T1/CDK9 totally abolished the association of vRNP with the C-terminal domain (CTD) Ser-2-phosphorylated form of RNA polymerase II. Further studies showed that overexpression of cyclin T1/CDK9 increased the transcription activity of vRNP, while knockdown of cyclin T1/CDK9 impaired viral replication. Our results suggest that cyclin T1/CDK9 serves as an adapter to mediate the interaction of vRNP and RNA Pol II and promote viral transcription.

scholarly journals Association of the Influenza A Virus RNA-Dependent RNA Polymerase with Cellular RNA Polymerase II

2005 ◽  
Vol 79 (9) ◽  
pp. 5812-5818 ◽  
Author(s):  
Othmar G. Engelhardt ◽  
Matt Smith ◽  
Ervin Fodor
Keyword(s):  
Influenza Virus ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Influenza A ◽  
Large Subunit ◽  
Viral Rna ◽  
Rna Dependent Rna Polymerase ◽  
Pol Ii ◽  
Virus Rna ◽  
Transcription Sites

ABSTRACT Transcription by the influenza virus RNA-dependent RNA polymerase is dependent on cellular RNA processing activities that are known to be associated with cellular RNA polymerase II (Pol II) transcription, namely, capping and splicing. Therefore, it had been hypothesized that transcription by the viral RNA polymerase and Pol II might be functionally linked. Here, we demonstrate for the first time that the influenza virus RNA polymerase complex interacts with the large subunit of Pol II via its C-terminal domain. The viral polymerase binds hyperphosphorylated forms of Pol II, indicating that it targets actively transcribing Pol II. In addition, immunofluorescence analysis is consistent with a new model showing that influenza virus polymerase accumulates at Pol II transcription sites. The present findings provide a framework for further studies to elucidate the mechanistic principles of transcription by a viral RNA polymerase and have implications for the regulation of Pol II activities in infected cells.

scholarly journals The Pol IV largest subunit CTD quantitatively affects siRNA levels guiding RNA-directed DNA methylation

2019 ◽  
Vol 47 (17) ◽  
pp. 9024-9036 ◽  
Author(s):  
Jered M Wendte ◽  
Jeremy R Haag ◽  
Olga M Pontes ◽  
Jasleen Singh ◽  
Sara Metcalf ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Catalytic Activity ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Cytosine Methylation ◽  
Transcriptional Gene Silencing ◽  
Rna Dependent Rna Polymerase ◽  
Pol Ii ◽  
Pol Iv ◽  
Non Coding Rnas

Abstract In plants, nuclear multisubunit RNA polymerases IV and V are RNA Polymerase II-related enzymes that synthesize non-coding RNAs for RNA-directed DNA methylation (RdDM) and transcriptional gene silencing. Here, we tested the importance of the C-terminal domain (CTD) of Pol IV’s largest subunit given that the Pol II CTD mediates multiple aspects of Pol II transcription. We show that the CTD is dispensable for Pol IV catalytic activity and Pol IV termination-dependent activation of RNA-DEPENDENT RNA POLYMERASE 2, which partners with Pol IV to generate dsRNA precursors of the 24 nt siRNAs that guide RdDM. However, 24 nt siRNA levels decrease ∼80% when the CTD is deleted. RNA-dependent cytosine methylation is also reduced, but only ∼20%, suggesting that siRNA levels typically exceed the levels needed for methylation of most loci. Pol IV-dependent loci affected by loss of the CTD are primarily located in chromosome arms, similar to loci dependent CLSY1/2 or SHH1, which are proteins implicated in Pol IV recruitment. However, deletion of the CTD does not phenocopy clsy or shh1 mutants, consistent with the CTD affecting post-recruitment aspects of Pol IV activity at target loci.

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 Sir2 Silences Gene Transcription by Targeting the Transition between RNA Polymerase II Initiation and Elongation

2008 ◽  
Vol 28 (12) ◽  
pp. 3979-3994 ◽  
Author(s):  
Lu Gao ◽  
David S. Gross
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Transcription Initiation ◽  
Transcriptional Silencing ◽  
Pol Ii ◽  
Initiation Factors ◽  
Lysine Deacetylase ◽  
Evolutionarily Conserved ◽  
Mrna Capping ◽  
Capping Enzyme

ABSTRACT It is well accepted that for transcriptional silencing in budding yeast, the evolutionarily conserved lysine deacetylase Sir2, in concert with its partner proteins Sir3 and Sir4, establishes a chromatin structure that prevents RNA polymerase II (Pol II) transcription. However, the mechanism of repression remains controversial. Here, we show that the recruitment of Pol II, as well as that of the general initiation factors TBP and TFIIH, occurs unimpeded to the silent HMR a 1 and HMLα1/HMLα2 mating promoters. This, together with the fact that Pol II is Ser5 phosphorylated, implies that SIR-mediated silencing is permissive to both preinitiation complex (PIC) assembly and transcription initiation. In contrast, the occupancy of factors critical to both mRNA capping and Pol II elongation, including Cet1, Abd1, Spt5, Paf1C, and TFIIS, is virtually abolished. In agreement with this, efficiency of silencing correlates not with a restriction in Pol II promoter occupancy but with a restriction in capping enzyme recruitment. These observations pinpoint the transition between polymerase initiation and elongation as the step targeted by Sir2 and indicate that transcriptional silencing is achieved through the differential accessibility of initiation and capping/elongation factors to chromatin. We compare Sir2-mediated transcriptional silencing to a second repression mechanism, mediated by Tup1. In contrast to Sir2, Tup1 prevents TBP, Pol II, and TFIIH recruitment to the HMLα1 promoter, thereby abrogating PIC formation.

scholarly journals A Nonconserved Surface of the TFIIB Zinc Ribbon Domain Plays a Direct Role in RNA Polymerase II Recruitment

2004 ◽  
Vol 24 (7) ◽  
pp. 2863-2874 ◽  
Author(s):  
Thomas C. Tubon ◽  
William P. Tansey ◽  
Winship Herr
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Transcription Initiation ◽  
Terminal Region ◽  
General Role ◽  
Pol Ii ◽  
Amino Terminal ◽  
Zinc Ribbon

ABSTRACT The general transcription factor TFIIB is a highly conserved and essential component of the eukaryotic RNA polymerase II (pol II) transcription initiation machinery. It consists of a single polypeptide with two conserved structural domains: an amino-terminal zinc ribbon structure (TFIIBZR) and a carboxy-terminal core (TFIIBCORE). We have analyzed the role of the amino-terminal region of human TFIIB in transcription in vivo and in vitro. We identified a small nonconserved surface of the TFIIBZR that is required for pol II transcription in vivo and for different types of basal pol II transcription in vitro. Consistent with a general role in transcription, this TFIIBZR surface is directly involved in the recruitment of pol II to a TATA box-containing promoter. Curiously, although the amino-terminal human TFIIBZR domain can recruit both human pol II and yeast (Saccharomyces cerevisiae) pol II, the yeast TFIIB amino-terminal region recruits yeast pol II but not human pol II. Thus, a critical process in transcription from many different promoters—pol II recruitment—has changed in sequence specificity during eukaryotic evolution.

scholarly journals The Myc Transactivation Domain Promotes Global Phosphorylation of the RNA Polymerase II Carboxy-Terminal Domain Independently of Direct DNA Binding

2007 ◽  
Vol 27 (6) ◽  
pp. 2059-2073 ◽  
Author(s):  
Victoria H. Cowling ◽  
Michael D. Cole
Keyword(s):  
Dna Binding ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Transcription Initiation ◽  
Target Genes ◽  
Dependent Manner ◽  
Transactivation Domain ◽  
Pol Ii ◽  
Terminal Domain ◽  
Ctd Phosphorylation

ABSTRACT Myc is a transcription factor which is dependent on its DNA binding domain for transcriptional regulation of target genes. Here, we report the surprising finding that Myc mutants devoid of direct DNA binding activity and Myc target gene regulation can rescue a substantial fraction of the growth defect in myc −/− fibroblasts. Expression of the Myc transactivation domain alone induces a transcription-independent elevation of the RNA polymerase II (Pol II) C-terminal domain (CTD) kinases cyclin-dependent kinase 7 (CDK7) and CDK9 and a global increase in CTD phosphorylation. The Myc transactivation domain binds to the transcription initiation sites of these promoters and stimulates TFIIH binding in an MBII-dependent manner. Expression of the Myc transactivation domain increases CDK mRNA cap methylation, polysome loading, and the rate of translation. We find that some traditional Myc transcriptional target genes are also regulated by this Myc-driven translation mechanism. We propose that Myc transactivation domain-driven RNA Pol II CTD phosphorylation has broad effects on both transcription and mRNA metabolism.

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 clustering through CTD phase separation

2018 ◽  
Author(s):  
M. Boehning ◽  
C. Dugast-Darzacq ◽  
M. Rankovic ◽  
A. S. Hansen ◽  
T. Yu ◽  
...  
Keyword(s):  
Phase Separation ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Transcription Initiation ◽  
Low Complexity ◽  
Initiation Factor ◽  
Published Data ◽  
Pol Ii ◽  
Intrinsically Disordered ◽  
Ctd Phosphorylation

The carboxy-terminal domain (CTD) of RNA polymerase (Pol) II is an intrinsically disordered low-complexity region that is critical for pre-mRNA transcription and processing. The CTD consists of hepta-amino acid repeats varying in number from 52 in humans to 26 in yeast. Here we report that human and yeast CTDs undergo cooperative liquid phase separation at increasing protein concentration, with the shorter yeast CTD forming less stable droplets. In human cells, truncation of the CTD to the length of the yeast CTD decreases Pol II clustering and chromatin association whereas CTD extension has the opposite effect. CTD droplets can incorporate intact Pol II and are dissolved by CTD phosphorylation with the transcription initiation factor IIH kinase CDK7. Together with published data, our results suggest that Pol II forms clusters/hubs at active genes through interactions between CTDs and with activators, and that CTD phosphorylation liberates Pol II enzymes from hubs for promoter escape and transcription elongation.

scholarly journals Association of the Influenza Virus RNA Polymerase Subunit PB2 with the Host Chaperonin CCT

2010 ◽  
Vol 84 (17) ◽  
pp. 8691-8699 ◽  
Author(s):  
Tatiana Fislová ◽  
Benjamin Thomas ◽  
Katy M. Graef ◽  
Ervin Fodor
Keyword(s):  
Influenza Virus ◽  
Rna Polymerase ◽  
Viral Replication ◽  
Mammalian Cells ◽  
Influenza A ◽  
Mitochondrial Protein ◽  
Host Factors ◽  
Fk506 Binding Protein ◽  
Virus Rna ◽  
Rna Accumulation

ABSTRACT The RNA polymerase of influenza A virus is a host range determinant and virulence factor. In particular, the PB2 subunit of the RNA polymerase has been implicated as a crucial factor that affects cell tropism as well as virulence in animal models. These findings suggest that host factors associating with the PB2 protein may play an important role during viral replication. In order to identify host factors that associate with the PB2 protein, we purified recombinant PB2 from transiently transfected mammalian cells and identified copurifying host proteins by mass spectrometry. We found that the PB2 protein associates with the cytosolic chaperonin containing TCP-1 (CCT), stress-induced phosphoprotein 1 (STIP1), FK506 binding protein 5 (FKBP5), α- and β-tubulin, Hsp60, and mitochondrial protein p32. Some of these binding partners associate with each other, suggesting that PB2 might interact with these proteins in multimeric complexes. More detailed analysis of the interaction of the PB2 protein with CCT revealed that PB2 associates with CCT as a monomer and that the CCT binding site is located in a central region of the PB2 protein. PB2 proteins from various influenza virus subtypes and origins can associate with CCT. Silencing of CCT resulted in reduced viral replication and reduced PB2 protein and viral RNA accumulation in a ribonucleoprotein reconstitution assay, suggesting an important function for CCT during the influenza virus life cycle. We propose that CCT might be acting as a chaperone for PB2 to aid its folding and possibly its incorporation into the trimeric RNA polymerase complex.

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.

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