Nascent elongating transcript sequencing (NET-seq) for Escherichia coli and Bacillus subtilis reveals a consensus pause sequence enriched at translation start sites.
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ABSTRACT:
PROVIDER: PRJNA244362 | ENA |
REPOSITORIES: ENA
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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
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.
2014-05-05 | GSE56720 | GEO
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
Project description:Transcriptional pausing is a fundamental mechanism that aids gene regulation by cellular RNA polymerases (RNAPs). In many bacterial lineages, a large, surface-exposed domain inserted into the catalytic trigger loop (TL) of RNAP called sequence insertion 3 (SI3) modulates transcription pausing. However, the in vivo roles of SI3 remain largely unknown due in part to the lethality of SI3 deletion. Here we describe construction of a viable Escherichia coli strain with a complete SI3 deletion enabled by a suppressor missense mutation in the portion of rpoC encoding the TL (ß′A941T; ∆SI3*). The ∆SI3* RNAP exhibited increased transcript elongation rate relative to ∆SI3 RNAP lacking the TL substitution, which may explain viability of the ∆SI3* strain. Using NET-seq, we found that transcriptional pausing in the ∆SI3* strain was increased at some sites and decreased at others compared to wild-type E. coli, except in ribosomal RNA genes. The ∆SI3*-enhanced pauses had a sequence motif similar to the consensus elemental pause sequence whereas ∆SI3*-suppressed pauses had less similarity to the consensus motif and appeared associated with upstream RNA structures thought to stabilize pausing (pause hairpins; PHs). These putative PH-stabilized pause signals were enriched in 5′ untranslated regions and protein coding regions in the E. coli genome. These results suggest that the SI3 domain affects different classes of pause signals differently and imply potential roles of these pauses in protein-coding regions.
2023-11-01 | GSE230757 | GEO
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
Project description: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.
2011-01-19 | GSE25107 | GEO
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
Project description:The advent of quantitative approaches that enable interrogation of transcription at single nucleotide resolution has allowed a novel understanding of transcriptional regulation previously undefined. To better map transcription genome-wide at base pair resolution and with transcription/elongation factor dependency we developed an adapted NET-seq protocol called NET-prism (Native Elongating Transcription by Polymerase-Regulated Immunoprecipitants in the Mammalian genome). NET-prism introduces an immunoprecipitation to capture RNA Pol II – associated proteins, which reveals the interaction of these proteins with active RNA Pol II. Application of NET-prism on different Pol II variants (Pol II S2ph, Pol II S5ph), elongation factors (Spt6, Ssrp1), splicing factors (Sf1), and components of the pre-initiation complex (PIC) (TFIID, and Mediator) reveals diverse Pol II signals, at a single nucleotide resolution, with regards to directionality and intensity over promoters, splice sites, and enhancers/super-enhancers. NET-prism will be broadly applicable as it exposes transcription factor/Pol II dependent topographic specificity and thus, a new degree of regulatory complexity.
2019-07-02 | GSE107257 | GEO
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
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