Global repression driven by RNA polymerase II stalling in yeast
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ABSTRACT:
PROVIDER: PRJNA635575 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR11862800_1.fastq.gz | Fastqsanger.gz | |||
| SRR11862800_2.fastq.gz | Fastqsanger.gz | |||
| SRR11862801_1.fastq.gz | Fastqsanger.gz | |||
| SRR11862801_2.fastq.gz | Fastqsanger.gz | |||
| SRR11862802_1.fastq.gz | Fastqsanger.gz |
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Project description:In multicellular eukaryotes, RNA polymerase (pol) II pauses transcription ~30-50 bp after initiation, and involves DSIF and other factors. While the budding yeast Saccharomyces has its transcription mechanisms mostly conserved with other eukaryotes, it appears to lack this fundamental promoter-proximal pausing. However, we now discover that acute stress (e.g., peroxide) causes most yeast genes, including constitutive and stress-induced genes, to manifest two distinct pausing or stall sites during the early elongation phase of Pol II transcription. The first stall occurs at the promoter, where Pol II melts the DNA and acquires all tested interactions normally. The second stall occurs 100-200 bp downstream. It too has most, but not all, of the tested interactions. This stall is phenocopied by depletion of DSIF. Our results suggest that similar to other eukaryotes, in yeast stalling globally down regulates nearly all Pol II transcription, but primarily in response to acute stimuli, whereas in other eukaryotes it represents the default state in all cells.
2021-01-20 | GSE151348 | GEO
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
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.
2014-04-21 | GSE54669 | GEO
Project description:In multicellular eukaryotes, RNA polymerase (Pol) II pauses transcription ~30-50 bp after initiation. While the budding yeast Saccharomyces has its transcription mechanisms mostly conserved with other eukaryotes, it appears to lack this fundamental promoter-proximal pausing. However, we now report that nearly all yeast genes, including constitutive and inducible genes, manifest two distinct transcriptional stall sites that are brought on by acute environmental signaling (e.g., peroxide stress). Pol II first stalls at the pre-initiation stage before promoter clearance, but after DNA melting and factor acquisition, and may involve inhibited dephosphorylation. The second stall occurs at the +2 nucleosome. It acquires most, but not all, elongation factor interactions. Its regulation may include Bur1/Spt4/5. Our results suggest that a double Pol II stall is a mechanism to downregulate essentially all genes in concert.
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Project description:We developed a novel approach to isolate and characterize, at nucleotide-level resolution, the RNAs derived from RNA polymerase II (Pol II) in early elongation complexes. We find that high numbers of these short RNAs are not correlated with gene expression but instead are indicative of promoter-proximally stalled polymerase. High amounts of these short RNAs are generated from more than one third of all genes, indicating that Pol II stalls in the promoter-proximal region on global scale. We also find that the nucleotide composition of the initially transcribed sequence plays an important role in promoting transcriptional stalling and that early elongation complexes are highly susceptible to backtracking and arrest. Global analysis of RNAs produced by promoter-proximal Pol II.
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Project description:Transcription-driven Chromatin Repression of Intragenic Promoters
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Project description:Short tandem repeats (STRs) significantly contribute to de novo mutagenesis, driving phenotypic diversity and genetic disease. Although highly diverse, their low complexity and repetitive nature induces DNA polymerase slippage and stalling, leading to length variation and base substitutions. However, the characterisation of DNA synthesis through STR loci has been restricted to a handful of selected sequences, limiting our broader understanding of their evolutionary behaviour. In order to understand the interplay between the ability of a given STR to impair DNA synthesis and its genomic stability, we developed a high-throughput polymerase extension assay that allows monitoring the kinetics of DNA synthesis at all STR permutations in different lengths in parallel. We have used the assay to map at single-nucleotide resolution the movement of a prototypical A-family replicative DNA polymerase (T7 DNA polymerase) through the repeats over time. From this data we can infer the secondary structure adopted by a given STR from the precise manner in which it stalls polymerase and link this to slippage and point mutation during DNA synthesis.
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Project description:Immunoglobulin enhancers increase RNA polymerase 2 stalling at somatic hypermutation target sequences
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Project description:Bacteria often evolve antibiotic resistance through mutagenesis. However, the processes causing the mutagenesis have not been fully resolved. Here we found that a broad range of ribosome-targeting antibiotics caused mutations through an underexplored pathway. Focusing on the clinically important aminoglycoside gentamicin, we found that the translation inhibitor caused genome-wide premature stalling of RNA polymerase (RNAP) in a loci-dependent manner. Further analysis showed that the stalling was caused by disruption of transcription-translation coupling. Anti-intuitively, the stalled RNAPs subsequently induced lesions to the DNA via transcription-coupled repair. While most of the bacteria were killed by genotoxicity, a small subpopulation acquired mutations via SOS-induced mutagenesis. Given that these processes were triggered shortly after antibiotic addition, resistance rapidly emerged in the population. Our work revealed a new mechanism of action of ribosomal antibiotics, illustrates the importance of dissecting the complex interplay between multiple molecular processes in understanding antibiotic efficacy, and suggests new strategies for countering the development of resistance.
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