scholarly journals Cdk9 regulates a promoter-proximal checkpoint to modulate RNA Polymerase II elongation rate

2017 ◽  
Author(s):  
Gregory T. Booth ◽  
Pabitra K. Parua ◽  
Miriam Sansó ◽  
Robert P. Fisher ◽  
John T. Lis
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Time Course ◽  
Kinase Activity ◽  
Elongation Rate ◽  
Transcription Start ◽  
Eukaryotic Transcription ◽  
Pol Ii ◽  
Cdk9 Kinase Activity

Multiple kinases modify RNA Polymerase II (Pol II) and its associated pausing and elongation factors to regulate Pol II transcription and transcription-coupled mRNA processing1,2. The conserved Cdk9 kinase is essential for regulated eukaryotic transcription3, but its mechanistic role remains incompletely understood. Here, we use altered-specificity kinase mutations and highly-specific inhibitors in fission yeast, Schizosaccharomyces pombe to examine the role of Cdk9, and related Cdk7 and Cdk12 kinases, on transcription at base-pair resolution using Precision Run-On sequencing (PRO-seq). Within a minute, Cdk9 inhibition causes a dramatic reduction in the phosphorylation of Pol II-associated factor, Spt5. The effects of Cdk9 inhibition on transcription are the more severe than inhibition of Cdk7 and Cdk12 and result in a shift of Pol II towards the transcription start site (TSS). A kinetic time course of Cdk9 inhibition reveals that early transcribing Pol II is the most compromised, with a measured rate of only ~400 bp/min, while Pol II that is already well into the gene continues rapidly to the end of genes with a rate > 1 kb/min. Our results indicate that while Pol II in S. pombe can escape promoter-proximal pausing in the absence of Cdk9 activity, it is impaired in elongation, suggesting the existence of a conserved global regulatory checkpoint that requires Cdk9 kinase activity.

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 Pausing sites of RNA polymerase II on actively transcribed genes are enriched in DNA double-stranded breaks

2020 ◽  
Vol 295 (12) ◽  
pp. 3990-4000 ◽  
Author(s):  
Sandeep Singh ◽  
Karol Szlachta ◽  
Arkadi Manukyan ◽  
Heather M. Raimer ◽  
Manikarna Dinda ◽  
...  
Keyword(s):  
Transcriptional Regulation ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Transcriptional Activation ◽  
Topoisomerase I ◽  
Cell Types ◽  
Dna Breaks ◽  
Transcription Start ◽  
Pol Ii ◽  
Transcription Start Sites

DNA double-stranded breaks (DSBs) are strongly associated with active transcription, and promoter-proximal pausing of RNA polymerase II (Pol II) is a critical step in transcriptional regulation. Mapping the distribution of DSBs along actively expressed genes and identifying the location of DSBs relative to pausing sites can provide mechanistic insights into transcriptional regulation. Using genome-wide DNA break mapping/sequencing techniques at single-nucleotide resolution in human cells, we found that DSBs are preferentially located around transcription start sites of highly transcribed and paused genes and that Pol II promoter-proximal pausing sites are enriched in DSBs. We observed that DSB frequency at pausing sites increases as the strength of pausing increases, regardless of whether the pausing sites are near or far from annotated transcription start sites. Inhibition of topoisomerase I and II by camptothecin and etoposide treatment, respectively, increased DSBs at the pausing sites as the concentrations of drugs increased, demonstrating the involvement of topoisomerases in DSB generation at the pausing sites. DNA breaks generated by topoisomerases are short-lived because of the religation activity of these enzymes, which these drugs inhibit; therefore, the observation of increased DSBs with increasing drug doses at pausing sites indicated active recruitment of topoisomerases to these sites. Furthermore, the enrichment and locations of DSBs at pausing sites were shared among different cell types, suggesting that Pol II promoter-proximal pausing is a common regulatory mechanism. Our findings support a model in which topoisomerases participate in Pol II promoter-proximal pausing and indicated that DSBs at pausing sites contribute to transcriptional activation.

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 Nucleosomal Fluctuations Govern the Transcription Dynamics of RNA Polymerase II

Science ◽  
2009 ◽  
Vol 325 (5940) ◽  
pp. 626-628 ◽  
Author(s):  
Courtney Hodges ◽  
Lacramioara Bintu ◽  
Lucyna Lubkowska ◽  
Mikhail Kashlev ◽  
Carlos Bustamante
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Optical Tweezers ◽  
Direct Evidence ◽  
Eukaryotic Transcription ◽  
Pol Ii ◽  
The Core ◽  
Nucleosomal Dna ◽  
Dna Loop ◽  
Core Histones

RNA polymerase II (Pol II) must overcome the barriers imposed by nucleosomes during transcription elongation. We have developed an optical tweezers assay to follow individual Pol II complexes as they transcribe nucleosomal DNA. Our results indicate that the nucleosome behaves as a fluctuating barrier that locally increases pause density, slows pause recovery, and reduces the apparent pause-free velocity of Pol II. The polymerase, rather than actively separating DNA from histones, functions instead as a ratchet that rectifies nucleosomal fluctuations. We also obtained direct evidence that transcription through a nucleosome involves transfer of the core histones behind the transcribing polymerase via a transient DNA loop. The interplay between polymerase dynamics and nucleosome fluctuations provides a physical basis for the regulation of eukaryotic transcription.

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 Ssl2/TFIIH function in transcription start site scanning by RNA Polymerase II in Saccharomyces cerevisiae

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Tingting Zhao ◽  
Irina O Vvedenskaya ◽  
William KM Lai ◽  
Shrabani Basu ◽  
B Franklin Pugh ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Core Promoter ◽  
Interaction Network ◽  
Transcription Start ◽  
Pol Ii ◽  
Transcription Start Sites ◽  
Scanning Rate ◽  
Residue Interaction

In Saccharomyces cerevisiae, RNA Polymerase II (Pol II) selects transcription start sites (TSS) by a unidirectional scanning process. During scanning, a preinitiation complex (PIC) assembled at an upstream core promoter initiates at select positions within a window ~40-120 basepairs downstream. Several lines of evidence indicate that Ssl2, the yeast homolog of XPB and an essential and conserved subunit of the general transcription factor (GTF) TFIIH, drives scanning through its DNA-dependent ATPase activity, therefore potentially controlling both scanning rate and scanning extent (processivity). To address questions of how Ssl2 functions in promoter scanning and interacts with other initiation activities, we leveraged distinct initiation-sensitive reporters to identify novel ssl2 alleles. These ssl2 alleles, many of which alter residues conserved from yeast to human, confer either upstream or downstream TSS shifts at the model promoter ADH1 and genome-wide. Specifically, tested ssl2 alleles alter TSS selection by increasing or narrowing the distribution of TSSs used at individual promoters. Genetic interactions of ssl2 alleles with other initiation factors are consistent with ssl2 allele classes functioning through increasing or decreasing scanning processivity but not necessarily scanning rate. These alleles underpin a residue interaction network that likely modulates Ssl2 activity and TFIIH function in promoter scanning. We propose that the outcome of promoter scanning is determined by two functional networks, the first being Pol II activity and factors that modulate it to determine initiation efficiency within a scanning window, and the second being Ssl2/TFIIH and factors that modulate scanning processivity to determine the width of the scanning widow.

scholarly journals Emerging Views on the CTD Code

2012 ◽  
Vol 2012 ◽  
pp. 1-19 ◽  
Author(s):  
David W. Zhang ◽  
Juan B. Rodríguez-Molina ◽  
Joshua R. Tietjen ◽  
Corey M. Nemec ◽  
Aseem Z. Ansari
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Transcription Regulation ◽  
Chromatin Remodeling ◽  
Posttranslational Modifications ◽  
Rna Processing ◽  
Transcription Initiation ◽  
Protein Complexes ◽  
Pol Ii

The C-terminal domain (CTD) of RNA polymerase II (Pol II) consists of conserved heptapeptide repeats that function as a binding platform for different protein complexes involved in transcription, RNA processing, export, and chromatin remodeling. The CTD repeats are subject to sequential waves of posttranslational modifications during specific stages of the transcription cycle. These patterned modifications have led to the postulation of the “CTD code” hypothesis, where stage-specific patterns define a spatiotemporal code that is recognized by the appropriate interacting partners. Here, we highlight the role of CTD modifications in directing transcription initiation, elongation, and termination. We examine the major readers, writers, and erasers of the CTD code and examine the relevance of describing patterns of posttranslational modifications as a “code.” Finally, we discuss major questions regarding the function of the newly discovered CTD modifications and the fundamental insights into transcription regulation that will necessarily emerge upon addressing those challenges.

scholarly journals Activator-Specific Requirement of Yeast Mediator Proteins for RNA Polymerase II Transcriptional Activation

1999 ◽  
Vol 19 (2) ◽  
pp. 979-988 ◽  
Author(s):  
Sang Jun Han ◽  
Young Chul Lee ◽  
Byung Soo Gim ◽  
Gi-Hyuck Ryu ◽  
Soon Jung Park ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Transcriptional Activation ◽  
Mediator Complex ◽  
Specific Requirement ◽  
Pol Ii ◽  
Mediator Protein ◽  
Mutant Forms ◽  
Mutant Cells

ABSTRACT The multisubunit Mediator complex of Saccharomyces cerevisiae is required for most RNA polymerase II (Pol II) transcription. The Mediator complex is composed of two subcomplexes, the Rgr1 and Srb4 subcomplexes, which appear to function in the reception of activator signals and the subsequent modulation of Pol II activity, respectively. In order to determine the precise composition of the Mediator complex and to explore the specific role of each Mediator protein, our goal was to identify all of the Mediator components. To this end, we cloned three previously unidentified Mediator subunits, Med9/Cse2, Med10/Nut2, and Med11, and isolated mutant forms of each of them to analyze their transcriptional defects. Differential display and Northern analyses of mRNAs from wild-type and Mediator mutant cells demonstrated an activator-specific requirement for each Mediator subunit. Med9/Cse2 and Med10/Nut2 were required, respectively, for Bas1/Bas2- and Gcn4-mediated transcription of amino acid biosynthetic genes. Gal11 was required for Gal4- and Rap1-mediated transcriptional activation. Med11 was also required specifically for MFα1 transcription. On the other hand, Med6 was required for all of these transcriptional activation processes. These results suggest that distinct Mediator proteins in the Rgr1 subcomplex are required for activator-specific transcriptional activation and that the activation signals mediated by these Mediator proteins converge on Med6 (or the Srb4 subcomplex) to modulate Pol II activity.

scholarly journals SETD5 modulates homeostasis of hematopoietic stem cells by mediating RNA Polymerase II pausing in cooperation with HCF-1

Leukemia ◽  
2021 ◽  
Author(s):  
Mengke Li ◽  
Chen Qiu ◽  
Yujie Bian ◽  
Deyang Shi ◽  
Bichen Wang ◽  
...  
Keyword(s):  
Stem Cells ◽  
Rna Polymerase ◽  
Hematopoietic Stem Cells ◽  
Rna Polymerase Ii ◽  
Critical Role ◽  
Whole Body ◽  
Hematopoietic Stem ◽  
Pol Ii

AbstractSETD5 mutations were identified as the genetic causes of neurodevelopmental disorders. While the whole-body knockout of Setd5 in mice leads to embryonic lethality, the role of SETD5 in adult stem cell remains unexplored. Here, a critical role of Setd5 in hematopoietic stem cells (HSCs) is identified. Specific deletion of Setd5 in hematopoietic system significantly increased the number of immunophenotypic HSCs by promoting HSC proliferation. Setd5-deficient HSCs exhibited impaired long-term self-renewal capacity and multiple-lineage differentiation potentials under transplantation pressure. Transcriptome analysis of Setd5-deficient HSCs revealed a disruption of quiescence state of long-term HSCs, a cause of the exhaustion of functional HSCs. Mechanistically, SETD5 was shown to regulate HSC quiescence by mediating the release of promoter-proximal paused RNA polymerase II (Pol II) on E2F targets in cooperation with HCF-1 and PAF1 complex. Taken together, these findings reveal an essential role of SETD5 in regulating Pol II pausing-mediated maintenance of adult stem cells.

scholarly journals RPAP2 regulates a transcription initiation checkpoint by prohibiting assembly of preinitiation complex

2021 ◽  
Author(s):  
Xizi Chen ◽  
Yilun Qi ◽  
Xinxin Wang ◽  
Zhenning Wang ◽  
Li Wang ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Phosphatase Activity ◽  
Transcription Initiation ◽  
Critical Role ◽  
In Vitro Transcription ◽  
Pol Ii ◽  
Activity Structure

RNA polymerase II (Pol II)-mediated transcription in metazoan requires precise regulation. RNA polymerase II-associated protein 2 (RPAP2) was previously identified to transport Pol II from cytoplasm to nucleus and dephosphorylates Pol II C-terminal domain (CTD). We found that RPAP2 binds hypo/hyper-phosphorylated Pol II with undetectable phosphatase activity. Structure of RPAP2-Pol II shows mutually exclusive assembly of RPAP2-Pol II and pre-initiation complex (PIC) due to three steric clashes. RPAP2 prevents/disrupts Pol II-TFIIF interaction and impairs in vitro transcription initiation, suggesting a function in prohibiting PIC assembly. Loss of RPAP2 in cells leads to global accumulation of TFIIF and Pol II at promoters, indicating critical role of RPAP2 in inhibiting PIC assembly independent of its putative phosphatase activity. Our study indicates that RPAP2 functions as a gatekeeper to prohibit PIC assembly and transcription initiation and suggests a novel transcription checkpoint.

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