scholarly journals Gene body H2B monoubiquitylation regulates gene-selective RNA Polymerase II pause release and is not rate limiting for transcription elongation

2015 ◽  
Author(s):  
Gilad Fuchs ◽  
Eran Rosenthal ◽  
Debora-Rosa Bublik ◽  
Tommy Kaplan ◽  
Moshe Oren
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Mammalian Cells ◽  
Molecular Mechanisms ◽  
Transcription Elongation ◽  
Causative Role ◽  
General Belief ◽  
Pol Ii ◽  
The Impact

Histone H2B monoubiquitylation (H2Bub1) is localized preferentially to transcribed regions of genes and spreads concomitantly with the progression of RNA polymerase II (Pol II). In mammalian cells, H2Bub1 levels are highly correlated with transcription elongation rates, consistent with the general belief that H2Bub1 facilitates the elongation process. Yet, a causative role of H2Bub1 in regulating elongation rates within live cells remains to be proven. Using our recently developed 4sUDRB-seq method, we examined the impact of H2Bub1 downregulation, through silencing of its cognate E3 ubiquitin ligase RNF20, on genomewide transcription elongation rates. Surprisingly, H2Bub1 downregulation had no measurable effect on global elongation rates. Instead, it led to upregulation of over 1,000 genes by altering their Pol II pause release times; notably, those genes are characterized by the presence of H2Bub1 in relatively close proximity to the paused Pol II. Conversely, another set of genes was downregulated upon partial H2Bub1 depletion, and in those genes H2Bub1 appeared to be required for efficient recruitment of Pol II to the promoter region. Overall, our data shed new light on the molecular mechanisms by which H2Bub1 regulates gene expression and imply that the role of H2Bub1 in transcription elongation should be reconsidered.

scholarly journals Rpb7 Can Interact with RNA Polymerase II and Support Transcription during Some Stresses Independently of Rpb4

1999 ◽  
Vol 19 (4) ◽  
pp. 2672-2680 ◽  
Author(s):  
Ayelet Sheffer ◽  
Mazal Varon ◽  
Mordechai Choder
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Stress Conditions ◽  
Cold Sensitivity ◽  
Starvation Period ◽  
Wild Type ◽  
Pol Ii ◽  
Growth Phenotype ◽  
Mild Temperature

ABSTRACT Rpb4 and Rpb7 are two yeast RNA polymerase II (Pol II) subunits whose mechanistic roles have recently started to be deciphered. Although previous data suggest that Rpb7 can stably interact with Pol II only as a heterodimer with Rpb4, RPB7 is essential for viability, whereas RPB4 is essential only during some stress conditions. To resolve this discrepancy and to gain a better understanding of the mode of action of Rpb4, we took advantage of the inability of cells lacking RPB4 (rpb4Δ, containing Pol IIΔ4) to grow above 30°C and screened for genes whose overexpression could suppress this defect. We thus discovered that overexpression of RPB7 could suppress the inability ofrpb4Δ cells to grow at 34°C (a relatively mild temperature stress) but not at higher temperatures. Overexpression ofRPB7 could also partially suppress the cold sensitivity ofrpb4Δ strains and fully suppress their inability to survive a long starvation period (stationary phase). Notably, however, overexpression of RPB4 could not override the requirement for RPB7. Consistent with the growth phenotype, overexpression of RPB7 could suppress the transcriptional defect characteristic of rpb4Δ cells during the mild, but not during a more severe, heat shock. We also demonstrated, through two reciprocal coimmunoprecipitation experiments, a stable interaction of the overproduced Rpb7 with Pol IIΔ4. Nevertheless, fewer Rpb7 molecules interacted with Pol IIΔ4 than with wild-type Pol II. Thus, a major role of Rpb4 is to augment the interaction of Rpb7 with Pol II. We suggest that Pol IIΔ4 contains a small amount of Rpb7 that is sufficient to support transcription only under nonstress conditions. When RPB7 is overexpressed, more Rpb7 assembles with Pol IIΔ4, enough to permit appropriate transcription also under some stress conditions.

scholarly journals Requirement for Yeast RAD26, a Homolog of the HumanCSB Gene, in Elongation by RNA Polymerase II

2001 ◽  
Vol 21 (24) ◽  
pp. 8651-8656 ◽  
Author(s):  
Sung-Keun Lee ◽  
Sung-Lim Yu ◽  
Louise Prakash ◽  
Satya Prakash
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Transcription Elongation ◽  
Rapid Synthesis ◽  
Developmental Defects ◽  
Developmental Abnormalities ◽  
Developmental Problems ◽  
Uv Lesions

ABSTRACT Mutations in the human CSB gene cause Cockayne syndrome (CS). In addition to increased photosensitivity, CS patients suffer from severe developmental abnormalities, including growth retardation and mental retardation. Whereas a deficiency in the preferential repair of UV lesions from the transcribed strand accounts for the increased photosensitivity of CS patients, the reason for developmental defects in these individuals has remained unclear. Here we provide in vivo evidence for a role of RAD26, the counterpart of the CSB gene in Saccharomyces cerevisiae, in transcription elongation by RNA polymerase II, and in addition we show that under conditions requiring rapid synthesis of new mRNAs, growth is considerably reduced in cells lackingRAD26. These findings implicate a role for CSB in transcription elongation, and they strongly suggest that impaired transcription elongation is the underlying cause of the developmental problems in CS patients.

scholarly journals The Role of RNA Polymerase II Elongation Control in HIV-1 Gene Expression, Replication, and Latency

2011 ◽  
Vol 2011 ◽  
pp. 1-9 ◽  
Author(s):  
Kyle A. Nilson ◽  
David H. Price
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Elongation Factor ◽  
Health Concern ◽  
Pol Ii ◽  
Positive Transcription Elongation Factor ◽  
Elongation Control ◽  
Hiv 1 ◽  
Latent Phase

HIV-1 usurps the RNA polymerase II elongation control machinery to regulate the expression of its genome during lytic and latent viral stages. After integration into the host genome, the HIV promoter within the long terminal repeat (LTR) is subject to potent downregulation in a postinitiation step of transcription. Once produced, the viral protein Tat commandeers the positive transcription elongation factor, P-TEFb, and brings it to the engaged RNA polymerase II (Pol II), leading to the production of viral proteins and genomic RNA. HIV can also enter a latent phase during which factors that regulate Pol II elongation may play a role in keeping the virus silent. HIV, the causative agent of AIDS, is a worldwide health concern. It is hoped that knowledge of the mechanisms regulating the expression of the HIV genome will lead to treatments and ultimately a cure.

scholarly journals RNA Polymerase II Accumulation in the Promoter-Proximal Region of the Dihydrofolate Reductase and γ-Actin Genes

2003 ◽  
Vol 23 (6) ◽  
pp. 1961-1967 ◽  
Author(s):  
Chonghui Cheng ◽  
Phillip A. Sharp
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Dihydrofolate Reductase ◽  
Mammalian Cells ◽  
Proximal Region ◽  
Heptad Repeat ◽  
Pol Ii ◽  
Actin Genes ◽  
Carboxyl Terminal ◽  
Promoter Proximal Region

ABSTRACT The carboxyl-terminal domain (CTD) of RNA polymerase II (Pol II) can be phosphorylated at serine 2 (Ser-2) and serine 5 (Ser-5) of the CTD heptad repeat YSPTSPS, and this phosphorylation is important in coupling transcription to RNA processing, including 5′ capping, splicing, and polyadenylation. The mammalian endogenous dihydrofolate reductase and γ-actin genes have been used to study the association of Pol II with different regions of transcribed genes (promoter-proximal compared to distal regions) and the phosphorylation status of its CTD. For both genes, Pol II is more concentrated in the promoter-proximal regions than in the interior regions. Moreover, different phosphorylation forms of Pol II are associated with distinct regions. Ser-5 phosphorylation of Pol II is concentrated near the promoter, while Ser-2 phosphorylation is observed throughout the gene. These results suggest that the accumulation of paused Pol II in promoter-proximal regions may be a common feature of gene regulation in mammalian cells.

Molecular mechanisms of RNA polymerase II transcription elongation elucidated by kinetic network models

2018 ◽  
Vol 49 ◽  
pp. 54-62 ◽  
Author(s):  
Ilona Christy Unarta ◽  
Lizhe Zhu ◽  
Carmen Ka Man Tse ◽  
Peter Pak-Hang Cheung ◽  
Jin Yu ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Molecular Mechanisms ◽  
Transcription Elongation ◽  
Network Models ◽  
Rna Polymerase Ii Transcription

scholarly journals Artificial Recruitment of TFIID, but Not RNA Polymerase II Holoenzyme, Activates Transcription in Mammalian Cells

2000 ◽  
Vol 20 (12) ◽  
pp. 4350-4358 ◽  
Author(s):  
David R. Dorris ◽  
Kevin Struhl
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Transcriptional Activation ◽  
Mammalian Cells ◽  
Yeast Cells ◽  
Pol Ii ◽  
Activation Domains ◽  
Synergistic Activation ◽  
The One ◽  
Rna Polymerase Ii Holoenzyme

ABSTRACT In yeast cells, transcriptional activation occurs when the RNA polymerase II (Pol II) machinery is artificially recruited to a promoter by fusing individual components of this machinery to a DNA-binding domain. Here, we show that artificial recruitment of components of the TFIID complex can activate transcription in mammalian cells. Surprisingly, artificial recruitment of TATA-binding protein (TBP) activates transiently transfected and chromosomally integrated promoters with equal efficiency, whereas artificial recruitment of TBP-associated factors activates only chromosomal reporters. In contrast, artificial recruitment of various components of the mammalian Pol II holoenzyme does not confer transcriptional activation, nor does it result in synergistic activation in combination with natural activation domains. In the one case examined in more detail, the Srb7 fusion failed to activate despite being associated with the Pol II holoenzyme and being directly recruited to the promoter. Interestingly, some acidic activation domains are less effective when the promoter is chromosomally integrated rather than transiently transfected, whereas the Sp1 glutamine-rich activation domain is more effective on integrated reporters. Thus, yeast and mammalian cells differ with respect to transcriptional activation by artificial recruitment of the Pol II holoenzyme.

scholarly journals Mechanism of DNA alkylation-induced transcriptional stalling, lesion bypass, and mutagenesis

2017 ◽  
Vol 114 (34) ◽  
pp. E7082-E7091 ◽  
Author(s):  
Liang Xu ◽  
Wei Wang ◽  
Jiabin Wu ◽  
Ji Hyun Shin ◽  
Pengcheng Wang ◽  
...  
Keyword(s):  
Rna Polymerase Ii ◽  
Molecular Mechanisms ◽  
Minor Groove ◽  
Dna Lesions ◽  
Dna Alkylation ◽  
Lesion Bypass ◽  
Pol Ii ◽  
The Impact

Alkylated DNA lesions, induced by both exogenous chemical agents and endogenous metabolites, interfere with the efficiency and accuracy of DNA replication and transcription. However, the molecular mechanisms of DNA alkylation-induced transcriptional stalling and mutagenesis remain unknown. In this study, we systematically investigated how RNA polymerase II (pol II) recognizes and bypasses regioisomeric O2-, N3-, and O4-ethylthymidine (O2-, N3-, and O4-EtdT) lesions. We observed distinct pol II stalling profiles for the three regioisomeric EtdT lesions. Intriguingly, pol II stalling at O2-EtdT and N3-EtdT sites is exacerbated by TFIIS-stimulated proofreading activity. Assessment for the impact of the EtdT lesions on individual fidelity checkpoints provided further mechanistic insights, where the transcriptional lesion bypass routes for the three EtdT lesions are controlled by distinct fidelity checkpoints. The error-free transcriptional lesion bypass route is strongly favored for the minor-groove O2-EtdT lesion. In contrast, a dominant error-prone route stemming from GMP misincorporation was observed for the major-groove O4-EtdT lesion. For the N3-EtdT lesion that disrupts base pairing, multiple transcriptional lesion bypass routes were found. Importantly, the results from the present in vitro transcriptional studies are well correlated with in vivo transcriptional mutagenesis analysis. Finally, we identified a minor-groove–sensing motif from pol II (termed Pro-Gate loop). The Pro-Gate loop faces toward the minor groove of RNA:DNA hybrid and is involved in modulating the translocation of minor-groove alkylated DNA template after nucleotide incorporation opposite the lesion. Taken together, this work provides important mechanistic insights into transcriptional stalling, lesion bypass, and mutagenesis of alkylated DNA lesions.

scholarly journals EWS, but Not EWS-FLI-1, Is Associated with Both TFIID and RNA Polymerase II: Interactions between Two Members of the TET Family, EWS and hTAFII68, and Subunits of TFIID and RNA Polymerase II Complexes

1998 ◽  
Vol 18 (3) ◽  
pp. 1489-1497 ◽  
Author(s):  
Anne Bertolotti ◽  
Thomas Melot ◽  
Joël Acker ◽  
Marc Vigneron ◽  
Olivier Delattre ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Chromosomal Translocation ◽  
Binding Studies ◽  
Pol Ii ◽  
Nuclear Extracts ◽  
Vitro Binding ◽  
Tfiid Complex

ABSTRACT The t(11;22) chromosomal translocation specifically linked to Ewing sarcoma and primitive neuroectodermal tumor results in a chimeric molecule fusing the amino-terminus-encoding region of theEWS gene to the carboxyl-terminal DNA-binding domain encoded by the FLI-1 gene. As the function of the protein encoded by the EWS gene remains unknown, we investigated the putative role of EWS in RNA polymerase II (Pol II) transcription by comparing its activity with that of its structural homolog, hTAFII68. We demonstrate that a portion of EWS is able to associate with the basal transcription factor TFIID, which is composed of the TATA-binding protein (TBP) and TBP-associated factors (TAFIIs). In vitro binding studies revealed that both EWS and hTAFII68 interact with the same TFIID subunits, suggesting that the presence of EWS and that of hTAFII68 in the same TFIID complex may be mutually exclusive. Moreover, EWS is not exclusively associated with TFIID but, similarly to hTAFII68, is also associated with the Pol II complex. The subunits of Pol II that interact with EWS and hTAFII68 have been identified, confirming the association with the polymerase. In contrast to EWS, the tumorigenic EWS–FLI-1 fusion protein is not associated with either TFIID or Pol II in Ewing cell nuclear extracts. These observations suggest that EWS and EWS–FLI-1 may play different roles in Pol II transcription.

scholarly journals Regulation of expression of human RNA polymerase II-transcribed snRNA genes

Open Biology ◽  
2017 ◽  
Vol 7 (6) ◽  
pp. 170073 ◽  
Author(s):  
Joana Guiro ◽  
Shona Murphy
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Molecular Mechanisms ◽  
Mrna Splicing ◽  
Regulation Of Expression ◽  
Protein Coding ◽  
Pol Ii ◽  
Protein Coding Genes ◽  
Non Coding Rnas ◽  
Rna Genes

In addition to protein-coding genes, RNA polymerase II (pol II) transcribes numerous genes for non-coding RNAs, including the small-nuclear (sn)RNA genes. snRNAs are an important class of non-coding RNAs, several of which are involved in pre-mRNA splicing. The molecular mechanisms underlying expression of human pol II-transcribed snRNA genes are less well characterized than for protein-coding genes and there are important differences in expression of these two gene types. Here, we review the DNA features and proteins required for efficient transcription of snRNA genes and co-transcriptional 3′ end formation of the transcripts.

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