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Native elongating transcript sequencing (NET-seq) of wild type Saccharomyces cerevisiae and of DST1, RCO1, SET1, SET2, EAF3 deletion strains

ABSTRACT: We present an approach (native elongating transcript sequencing, NET-seq), based on deep sequencing of 3’ ends of nascent transcripts associated with RNA polymerase, to monitor transcription at nucleotide resolution. Application of NET-seq in Saccharomyces cerevisiae reveals that while promoters are generally capable of divergent transcription, the Rpd3S deacetylation complex enforces strong directionality to most promoters by suppressing antisense transcript initiation. Our studies also reveal pervasive polymerase pausing and backtracking throughout the body of transcripts. Average pause density shows prominent peaks at each of the first four nucleosomes, with the peak location occurring in good agreement with in vitro biophysical measurements. Thus nucleosome-induced pausing represents a major barrier to transcriptional elongation in vivo.

ORGANISM(S): Saccharomyces cerevisiae

PROVIDER: GSE25107 | GEO | 2011/01/19

SECONDARY ACCESSION(S): PRJNA134499

REPOSITORIES: GEO

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Native elongating transcript sequencing (NET-seq) of wild type Saccharomyces cerevisiae and of DST1, RCO1, SET1, SET2, EAF3 deletion strains

Project description:We present an approach (native elongating transcript sequencing, NET-seq), based on deep sequencing of 3M-bM-^@M-^Y ends of nascent transcripts associated with RNA polymerase, to monitor transcription at nucleotide resolution. Application of NET-seq in Saccharomyces cerevisiae reveals that while promoters are generally capable of divergent transcription, the Rpd3S deacetylation complex enforces strong directionality to most promoters by suppressing antisense transcript initiation. Our studies also reveal pervasive polymerase pausing and backtracking throughout the body of transcripts. Average pause density shows prominent peaks at each of the first four nucleosomes, with the peak location occurring in good agreement with in vitro biophysical measurements. Thus nucleosome-induced pausing represents a major barrier to transcriptional elongation in vivo. Examination of nascent transcripts in yeast and mutant strains

2011-01-19 | E-GEOD-25107 | biostudies-arrayexpress

Nascent elongating transcript sequencing (NET-seq) for Escherichia coli and Bacillus subtilis reveals a consensus pause sequence enriched at translation start sites.

Project description:Transcription by RNA polymerase (RNAP) is interrupted by pauses that play diverse regulatory roles. Although individual pauses have been studied in vitro, the determinants of pauses in vivo and their distribution throughout the bacterial genome remain unknown. Using nascent transcript sequencing we identify a 16 nt consensus pause sequence in E. coli that accounts for known regulatory pause sites as well as ~20,000 new in vivo pause sites. In vitro single-molecule and ensemble analyses demonstrate that these pauses result from RNAP/nucleic-acid interactions that inhibit next-nucleotide addition. The consensus sequence also leads to pausing by RNAPs from diverse lineages and is enriched at translation start sites in both E. coli and B. subtilis. Our results thus implicate a conserved mechanism unifying known and newly identified pause events. Examination of nascent transcripts in E. coli and B. subtilis. 6 samples of E. coli NET-seq, 1 sample of E. coli mRNA-seq, and 1 sample of B. subtilis NET-seq.

2014-05-05 | E-GEOD-56720 | biostudies-arrayexpress

TEF-seq/NET-seq applied to factors Rpb3, Paf1, Spt6, Spt16, Cet1, Pcf11, Ssu72, Set2

Project description:Here we adapt native elongating transcript sequencing (NET-seq) to develop transcription elongation factor associated nascent elongating transcript sequencing (TEF-seq). In this the RNA polymerase II (Pol2) transcription elongation complex (TEC) is immunoprecipitated via associated transcription elongation factors (TEFs). Sequencing from the 3' end of the Pol2-associated nascent transcript shows the position of the final incorporated nucleotide giving strand-specific, single nucleotide resolution maps of the level of TEF association with Pol2 during transcription.

2021-01-18 | E-MTAB-4568 | biostudies-arrayexpress

Nascent elongating transcript sequencing (NET-seq) show different pause registers with apyrase treatment during library preparation

Project description:Transcriptional pausing aids gene regulation by cellular RNA polymerases (RNAPs). In many bacteria, a surface-exposed domain inserted into the catalytic trigger loop (TL) of RNAP, called SI3 in Escherichia coli, modulates pausing and is essential for growth. Here we describe a viable E. coli strain lacking SI3 enabled by a suppressor TL substitution (β'Ala941→Thr; ∆SI3*). ∆SI3* increased transcription rate in vitro relative to ∆SI3, possibly explaining its viability, but retained both positive and negative effects of ∆SI3 on pausing. ∆SI3* inhibited pauses stabilized by nascent RNA structures (pause hairpins; PHs) but enhanced other pauses. Using NET-seq, we found that ∆SI3*-enhanced pauses resemble the consensus elemental pause sequence whereas sequences at ∆SI3*-suppressed pauses, which exhibited greater association with PHs, were more divergent. ∆SI3*-suppressed pauses also were associated with apparent pausing one nt upstream from the consensus sequence, often generating tandem pause sites. These '–2 pauses' were stimulated by pyrophosphate in vitro and by addition of apyrase to degrade residual NTPs during NET-seq sample processing. We propose that some pauses are readily reversible by pyrophosphorolysis or single-nucleotide cleavage. Our results document multiple ways that SI3 modulates pausing in vivo and may explain discrepancies in consensus pause sequences in some NET-seq studies.

2023-11-01 | GSE240163 | GEO

Native Elongating Transcript Sequencing Reveals Human Transcriptional Activity at Nucleotide Resolution

Project description:Major features of transcription by human RNA polymerase II (Pol II) remain poorly defined due to a lack of quantitative approaches for visualizing Pol II progress at nucleotide resolution. We developed a simple and powerful approach for performing native elongating transcript sequencing (NET-seq) in human cells that globally maps strand-specific Pol II density at nucleotide resolution. NET-seq exposes a mode of antisense transcription that originates downstream and converges on transcription from the canonical promoter. Convergent transcription is associated with a distinctive chromatin configuration and is characteristic of lower-expressed genes. Integration of NET-seq with genomic footprinting data reveals stereotypic Pol II pausing coincident with transcription factor occupancy. Finally, exons retained in mature transcripts display Pol II pausing signatures that differ markedly from skipped exons, indicating an intrinsic capacity for Pol II to recognize exons with different processing fates.

2015-04-23 | GSE61332 | GEO

Native Elongating Transcript sequencing (NET-seq) in wild-type and three members of the CAF-I complex.

Project description:We performed a fluorescent reporter based screen to identify factors determining transcriptional directionality from bidirectional promoters that give rise to a coding and a non-coding transcript. Promoters like these are most frequent in many organisms and non-coding transcription from this origin represents a large fraction of total long non-coding transcripts. We applied NET-seq to compare nascent transcription in yeast wild-type and mutations in the three members of the CAF-I complex. This technique avoids the technical issue of co-transcriptional RNA degradation and is a powerful approach to address questions concerning non-coding transcription. We find divergent non-coding transcription to be elevated in CAF-I mutants. One condition, yeast grown in YPD media to mid-log phase. 8 NET-seq libraries are deposited, 2 biological repeats of the wild-type and two biological repeast each of the CAF-I complex member mutants (CAC1, CAC2, CAC3).

2014-06-01 | E-GEOD-55982 | biostudies-arrayexpress

set2 buratowski lab

Project description:H3K36 methylation by Set2 targets Rpd3S histone deacetylase to 3Ã¢ÂÂ transcribed regions, repressing internal cryptic promoters and slowing elongation. To further explore the function of this pathway, global transcription was analyzed in yeast experiencing a series of carbon source shifts. Many previously unreported cryptic ncRNAs are induced by specific carbon sources, showing that cryptic promoters can be environmentally regulated. Also, approximately 230 mRNA genes show altered induction or repression upon SET2 deletion. A majority of Set2-repressed genes have overlapping lncRNA transcription. At these promoters, H3K36me3 and deacetylation by Rpd3S leads to slower or reduced induction. Unexpectedly, Set2 derepresses 159 genes, apparently by mediating a balance between Rpd3S and Rpd3L. Therefore, in addition to repression of cryptic transcription, H3K36 methylation maintains optimal expression dynamics of many mRNAs.

2016-09-25 | E-MTAB-4268 | biostudies-arrayexpress

DksA Guards Elongating RNA Polymerase Against Ribosome-Stalling-Induced Arrest

2014-04-21 | GSE54669 | GEO

DksA Guards Elongating RNA Polymerase Against Ribosome-Stalling-Induced Arrest

Project description:In bacteria, translation-transcription coupling inhibits RNA polymerase (RNAP) stalling. We present evidence suggesting that, upon amino acid starvation, inactive ribosomes promote rather than inhibit RNAP stalling. We developed an algorithm to evaluate genome-wide polymerase progression independently of local noise, and used it to reveal that the transcription factor DksA inhibits promoter-proximal pausing and increases RNAP elongation when uncoupled from translation by depletion of charged tRNAs. DksA has minimal effect on RNAP elongation in vitro and on untranslated RNAs in vivo. In these cases, transcripts can form RNA structures that prevent backtracking. Thus, the effect of DksA on transcript elongation may occur primarily upon ribosome slowing/stalling or at promoter-proximal locations that limit the potential for RNA structure. We propose that inactive ribosomes prevent formation of backtrackblocking mRNA structures and that, in this circumstance, DksA acts as a transcription elongation factor in vivo. Chromatin immunoprecipitation (ChIP) experiments were performed by using antibodies against RNA polymerase b subunit in wild-type and DdksA cells treated with 0.5mg/ml serine hydroxamate (SHX) or untreated. DksA and s70 enrichments were compared to RNAP enrichment by ChIP experiments using antibodies against s70 and DksA in wild-type cells (also in DdksA cells as a negative control for DksA ChIP-chip). Differentially labeled ChIP DNA and genomic DNA were competitively hybridized to an E. coli K-12 MG1655 tiling array with overlapping probes at ~12bp spacing across the entire genome. The series contains 19 datasets.

2014-04-21 | E-GEOD-54669 | biostudies-arrayexpress

Native Elongating Transcript sequencing (NET-seq) in wild-type and three members of the CAF-I complex

2014-06-01 | GSE55982 | GEO

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