Control of Transcriptional Elongation by RNA Polymerase II: A Retrospective

The origins of our current understanding of control of transcription elongation lie in pioneering experiments that mapped RNA polymerase II on viral and cellular genes. These studies first uncovered the surprising excess of polymerase molecules that we now know to be situated at the at the 5′ ends of most genes in multicellular organisms. The pileup of pol II near transcription start sites reflects a ubiquitous bottle-neck that limits elongation right at the start of the transcription elongation. Subsequent seminal work identified conserved protein factors that positively and negatively control the flux of polymerase through this bottle-neck, and make a major contribution to control of gene expression.

Download Full-text

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

Journal of Biological Chemistry ◽

10.1074/jbc.ra119.011665 ◽

2020 ◽

Vol 295 (12) ◽

pp. 3990-4000 ◽

Cited By ~ 3

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.

Download Full-text

RNA polymerase II-independent recruitment of SPT6L at transcription start sites in Arabidopsis

Nucleic Acids Research ◽

10.1093/nar/gkz465 ◽

2019 ◽

Vol 47 (13) ◽

pp. 6714-6725 ◽

Cited By ~ 3

Author(s):

Chen Chen ◽

Jie Shu ◽

Chenlong Li ◽

Raj K Thapa ◽

Vi Nguyen ◽

...

Keyword(s):

Rna Polymerase ◽

Rna Polymerase Ii ◽

Transcription Initiation ◽

Elongation Factor ◽

Proper Function ◽

Gene Body ◽

Transcription Start ◽

Transcription Start Sites ◽

Genome Wide ◽

Yeast Mutants

Abstract SPT6 is a conserved elongation factor that is associated with phosphorylated RNA polymerase II (RNAPII) during transcription. Recent transcriptome analysis in yeast mutants revealed its potential role in the control of transcription initiation at genic promoters. However, the mechanism by which this is achieved and how this is linked to elongation remains to be elucidated. Here, we present the genome-wide occupancy of Arabidopsis SPT6-like (SPT6L) and demonstrate its conserved role in facilitating RNAPII occupancy across transcribed genes. We also further demonstrate that SPT6L enrichment is unexpectedly shifted, from gene body to transcription start site (TSS), when its association with RNAPII is disrupted. Protein domains, required for proper function and enrichment of SPT6L on chromatin, are subsequently identified. Finally, our results suggest that recruitment of SPT6L at TSS is indispensable for its spreading along the gene body during transcription. These findings provide new insights into the mechanisms underlying SPT6L recruitment in transcription and shed light on the coordination between transcription initiation and elongation.

Download Full-text

CUT&RUN detects distinct DNA footprints of RNA polymerase II near the transcription start sites

Chromosome Research ◽

10.1007/s10577-020-09643-0 ◽

2020 ◽

Vol 28 (3-4) ◽

pp. 381-393

Author(s):

Michi Miura ◽

Honglin Chen

Keyword(s):

Rna Polymerase ◽

Rna Polymerase Ii ◽

Chromatin Immunoprecipitation ◽

Biological Significance ◽

Micrococcal Nuclease ◽

Dna Fragments ◽

Transcription Start ◽

Transcription Start Sites ◽

Human Lung Carcinoma Cell

AbstractCUT&RUN is a powerful tool to study protein-DNA interactions in vivo. DNA fragments cleaved by the targeted micrococcal nuclease identify the footprints of DNA-binding proteins on the chromatin. We performed CUT&RUN on human lung carcinoma cell line A549 maintained in a multi-well cell culture plate to profile RNA polymerase II. Long (> 270 bp) DNA fragments released by CUT&RUN corresponded to the bimodal peak around the transcription start sites, as previously seen with chromatin immunoprecipitation. However, we found that short (< 120 bp) fragments identify a well-defined peak localised at the transcription start sites. This distinct DNA footprint of short fragments, which constituted only about 5% of the total reads, suggests the transient positioning of RNA polymerase II before promoter-proximal pausing, which has not been detected in the physiological settings by standard chromatin immunoprecipitation. We showed that the positioning of the large-size-class DNA footprints around the short-fragment peak was associated with the directionality of transcription, demonstrating the biological significance of distinct CUT&RUN footprints of RNA polymerase II.

Download Full-text

Properties of an Intergenic Terminator and Start Site Switch That Regulate IMD2 Transcription in Yeast

Molecular and Cellular Biology ◽

10.1128/mcb.00380-08 ◽

2008 ◽

Vol 28 (12) ◽

pp. 3883-3893 ◽

Cited By ~ 60

Author(s):

M. Harley Jenks ◽

Thomas W. O'Rourke ◽

Daniel Reines

Keyword(s):

Rna Polymerase ◽

Rna Polymerase Ii ◽

Initiation Site ◽

Start Codon ◽

Start Site ◽

Transcription Start ◽

Transcription Start Sites ◽

Short Transcript ◽

Alternative Transcription ◽

Polyadenylation Sites

ABSTRACT The IMD2 gene in Saccharomyces cerevisiae is regulated by intracellular guanine nucleotides. Regulation is exerted through the choice of alternative transcription start sites that results in synthesis of either an unstable short transcript terminating upstream of the start codon or a full-length productive IMD2 mRNA. Start site selection is dictated by the intracellular guanine nucleotide levels. Here we have mapped the polyadenylation sites of the upstream, unstable short transcripts that form a heterogeneous family of RNAs of ≈200 nucleotides. The switch from the upstream to downstream start sites required the Rpb9 subunit of RNA polymerase II. The enzyme's ability to locate the downstream initiation site decreased exponentially as the start was moved downstream from the TATA box. This suggests that RNA polymerase II's pincer grip is important as it slides on DNA in search of a start site. Exosome degradation of the upstream transcripts was highly dependent upon the distance between the terminator and promoter. Similarly, termination was dependent upon the Sen1 helicase when close to the promoter. These findings extend the emerging concept that distinct modes of termination by RNA polymerase II exist and that the distance of the terminator from the promoter, as well as its sequence, is important for the pathway chosen.

Download Full-text

CUT&RUN detects distinct DNA footprints of RNA polymerase II near the transcription start sites

10.1101/2020.07.07.191478 ◽

2020 ◽

Author(s):

Michi Miura ◽

Honglin Chen

Keyword(s):

Rna Polymerase ◽

Rna Polymerase Ii ◽

Chromatin Immunoprecipitation ◽

Biological Significance ◽

Micrococcal Nuclease ◽

Dna Fragments ◽

Transcription Start ◽

Transcription Start Sites ◽

Human Lung Carcinoma Cell

AbstractCUT&RUN is a powerful tool to study protein-DNA interactions in vivo. DNA fragments cleaved by the targeted micrococcal nuclease identify the footprints of DNA-binding proteins on the chromatin. We performed CUT&RUN on human lung carcinoma cell line A549 maintained in a multi-well cell culture plate to profile RNA polymerase II. Long (>270 bp) DNA fragments released by CUT&RUN corresponded to the bimodal peak around the transcription start sites, as previously seen with chromatin immunoprecipitation. However, we found that short (<120 bp) fragments identify a well-defined peak localised at the transcription start sites. This distinct DNA footprint of short fragments, which constituted only about 5% of the total reads, suggests the transient positioning of RNA polymerase II before promoter-proximal pausing, which has not been detected in the physiological settings by standard chromatin immunoprecipitation. We showed that the positioning of the large-size-class DNA footprints around the short-fragment peak was associated with the directionality of transcription, demonstrating the biological significance of distinct CUT&RUN footprints of RNA polymerase II.

Download Full-text

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

eLife ◽

10.7554/elife.71013 ◽

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.

Download Full-text

Novel tools for the prediction of promoters in plants and bacteria

Faktori eksperimental'noi evolucii organizmiv ◽

10.7124/feeo.v20.793 ◽

1970 ◽

pp. 351-355

Author(s):

I. A. Shahmuradov

Keyword(s):

Rna Polymerase ◽

Rna Polymerase Ii ◽

Sigma Factors ◽

Promoter Prediction ◽

Transcription Start ◽

Transcription Start Sites ◽

Computer Tools ◽

E Coli ◽

Prediction Program ◽

Rna Polymerase Promoter

Aim. The computational search for promoters remains an attractive problem in bioinformatics. Despite the attention it has received for many years, the problem has not been addressed satisfactorily. These studies were aimed to develop novel computer tools for prediction of promoters (transcription start sites, TSSs) in plants and bacteria. Results. Two novel tools for prediction of RNA polymerase II promoters in plants (TSSPlant) and bacteria (bTSSfinder) have been developed. TSSPlant achieves significantly higher accuracy compared to the next best promoter prediction program for both TATA and TATA-less promoters; it is available to download as a standalone program at http://www.cbrc.kaust.edu.sa/download/. bTSSfinder predicts promoters for five classes of σ factors in Cyanobacteria (σA, σC, σH, σG and σF) and for five classes of sigma factors in E. coli (σ70, σ38, σ32, σ28 and σ24). Comparing to currently available tools, bTSSfinder achieves highest accuracy. bTSSfinder is available standalone and online at http://www.cbrc.kaust.edu.sa/btssfinder. Conclusions. To date, TSSPlant and bTSSfinder are most accurate promoter predictors in plants and bacteria, respectively. Keywords: transcription, RNA polymerase, promoter, TSS, promoter prediction.

Download Full-text

Phosphorylation of the RNA Polymerase II Carboxyl-Terminal Domain by CDK9 Is Directly Responsible for Human Immunodeficiency Virus Type 1 Tat-Activated Transcriptional Elongation

Molecular and Cellular Biology ◽

10.1128/mcb.22.13.4622-4637.2002 ◽

2002 ◽

Vol 22 (13) ◽

pp. 4622-4637 ◽

Cited By ~ 103

Author(s):

Young Kyeung Kim ◽

Cyril F. Bourgeois ◽

Catherine Isel ◽

Mark J. Churcher ◽

Jonathan Karn

Keyword(s):

Human Immunodeficiency Virus ◽

Rna Polymerase ◽

Rna Polymerase Ii ◽

Transcription Elongation ◽

Transcriptional Elongation ◽

Tat Protein ◽

Immunodeficiency Virus ◽

Carboxyl Terminal ◽

Transcription Complexes

ABSTRACT Stimulation of transcriptional elongation by the human immunodeficiency virus type 1 Tat protein is mediated by CDK9, a kinase that phosphorylates the RNA polymerase II carboxyl-terminal domain (CTD). In order to obtain direct evidence that this phosphorylation event can alter RNA polymerase processivity, we prepared transcription elongation complexes that were arrested by the lac repressor. The CTD was then dephosphorylated by treatment with protein phosphatase 1. The dephosphorylated transcription complexes were able to resume the transcription elongation when IPTG (isopropyl-β-d-thiogalactopyranoside) and nucleotides were added to the reaction. Under these chase conditions, efficient rephosphorylation of the CTD was observed in complexes containing the Tat protein but not in transcription complexes prepared in the absence of Tat protein. Immunoblots and kinase assays with synthetic peptides showed that Tat activated CDK9 directly since the enzyme and its cyclin partner, cyclin T1, were present at equivalent levels in transcription complexes prepared in the presence or absence of Tat. Chase experiments with the dephosphorylated elongation transcription complexes were performed in the presence of the CDK9 kinase inhibitor DRB (5,6-dichloro-1-β-d-ribofuranosyl-benzimidazole). Under these conditions there was no rephosphorylation of the CTD during elongation, and transcription through either a stem-loop terminator or bent DNA arrest sequence was strongly inhibited. In experiments in which the CTD was phosphorylated prior to elongation, the amount of readthrough of the terminator sequences was proportional to the extent of the CTD modification. The change in processivity is due to CTD phosphorylation alone, since even after the removal of Spt5, the second substrate for CDK9, RNA polymerase elongation is enhanced by Tat-activated CDK9 activity. We conclude that phosphorylation of the RNA polymerase II CTD by CDK9 enhances transcription elongation directly.

Download Full-text

Elongin B-Mediated Epigenetic Alteration of Viral Chromatin Correlates with Efficient Human Cytomegalovirus Gene Expression and Replication

mBio ◽

10.1128/mbio.00023-11 ◽

2011 ◽

Vol 2 (2) ◽

Cited By ~ 14

Author(s):

Jiwon Hwang ◽

Ryan T. Saffert ◽

Robert F. Kalejta

Keyword(s):

Gene Expression ◽

Rna Polymerase ◽

Rna Polymerase Ii ◽

Human Cytomegalovirus ◽

Viral Gene ◽

Epigenetic Mark ◽

Protein Accumulation ◽

Transcriptional Elongation ◽

Histone H2b ◽

Cellular Genes

ABSTRACTElongins B and C are members of complexes that increase the efficiency of transcriptional elongation by RNA polymerase II (RNAPII) and enhance the monoubiquitination of histone H2B, an epigenetic mark of actively transcribed genes. Here we show that, in addition to its role in facilitating transcription of the cellular genome, elongin B also enhances gene expression from the double-stranded DNA genome of human cytomegalovirus (HCMV), a pathogenic herpesvirus. Reducing the level of elongin B by small interfering RNA- or short hairpin RNA-mediated knockdown decreased viral mRNA expression, viral protein accumulation, viral DNA replication, and infectious virion production. Chromatin immunoprecipitation analysis indicated viral genome occupancy of the elongating form of RNAPII, and monoubiquitinated histone H2B was reduced in elongin B-deficient cells. These data suggest that, in addition to the previously documented epigenetic regulation of transcriptional initiation, HCMV also subverts cellular elongin B-mediated epigenetic mechanisms for enhancing transcriptional elongation to enhance viral gene expression and virus replication.IMPORTANCEThe genetic and epigenetic control of transcription initiation at both cellular and viral promoters is well documented. Recently, the epigenetic modification of histone H2B monoubiquitination throughout the bodies of cellular genes has been shown to enhance the elongation of RNA polymerase II-initiated transcripts. Mechanisms that might control the elongation of viral transcripts are less well studied. Here we show that, as with cellular genes, elongin B-mediated monoubiquitination of histone H2B also facilitates the transcriptional elongation of human cytomegalovirus genes. This and perhaps other epigenetic markings of actively transcribed regions may help in identifying viral genes expressed duringin vitrolatency or during natural infections of humans. Furthermore, this work identifies a novel, tractable model system to further study the regulation of transcriptional elongation in living cells.

Download Full-text