An allosteric/intrinsic mechanism supports termination of snRNA transcription.
Ontology highlight
ABSTRACT:
PROVIDER: PRJNA631616 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR11749628.fastq.gz | Fastqsanger.gz | |||
| SRR11749629.fastq.gz | Fastqsanger.gz | |||
| SRR11749630.fastq.gz | Fastqsanger.gz | |||
| SRR11749631.fastq.gz | Fastqsanger.gz | |||
| SRR11749632.fastq.gz | Fastqsanger.gz |
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Project description:Termination of protein-coding gene transcription by RNA polymerase II (Pol II) depends on endonucleolytic cleavage at the poly(A) site and the activity of a 5’->3’ “torpedo” exoribonuclease. Other Pol II transcripts also undergo endonucleolytic cleavage suggesting common themes for its termination. Nevertheless, many RNA polymerases employ intrinsic/allosteric termination that directly defines transcript 3’ ends. We have analysed termination of snRNA transcription in humans, which utilises the endoribonucleolytic integrator complex. Integrator is involved, but termination continues after its depletion. This implicates additional mechanisms and shows that endo- and 5’->3’ exonuclease activities are not necessarily a basis for all Pol II termination. Although the alternative process acts as a failsafe in the absence of integrator it contributes significantly to snRNA termination in the natural situation. Long-read sequencing reveals its products to have stochastic 3’ ends that are terminated before integrator cleavage, ruling out 5’->3’ exonuclease contribution(s) and supporting an allosteric/intrinsic mechanism. Termination of some snRNA transcription occurs at T-runs further indicating common principles between this mechanism and those used by other RNA polymerases.
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Project description:The so-called allosteric and torpedo models have been used for the past thirty years to explain how transcription terminates on protein-coding genes. The former invokes conformational changes in the transcription complex and the latter involves degradation of the downstream product of poly(A) signal (PAS) processing. Here, we describe a single mechanism incorporating features of both models. We show that CPSF73 is indispensable for transcriptional termination on protein-coding and its loss causes profound read-through genome-wide. CPSF73 functions upstream of allosteric modifications to the elongation complex that cause Pol II to slow down after the PAS. This state is enriched by rapid depletion of XRN2 and promoted by protein phosphatase 1 (PP1), the inhibition of which confers runaway read-through in the absence of XRN2. These allosteric changes facilitate XRN2-dependent termination, by aiding its capture of Pol II, rather than constituting a termination pathway in themselves. Our experiments unify the long-standing allosteric and torpedo models for transcriptional termination.
2020-01-21 | GSE137727 | GEO
Project description:Many RNA polymerases terminate transcription using allosteric/intrinsic mechanisms, whereby protein alterations or nucleotide sequences promote their release from DNA. RNA polymerase II (Pol II) is somewhat different based on its behavior at protein-coding genes where termination additionally requires endoribonucleolytic cleavage and subsequent 5'→3' exoribonuclease activity. The Pol-II-transcribed small nuclear RNAs (snRNAs) also undergo endoribonucleolytic cleavage by the Integrator complex, which promotes their transcriptional termination. Here, we confirm the involvement of Integrator but show that Integrator-independent processes can terminate snRNA transcription both in its absence and naturally. This is often associated with exosome degradation of snRNA precursors that long-read sequencing analysis reveals as frequently terminating at T-runs located downstream of some snRNAs. This finding suggests a unifying vulnerability of RNA polymerases to such sequences given their well-known roles in terminating Pol III and bacterial RNA polymerase.
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Project description:In this work we conducted Term-seq on Wild Type B. subtilis, and strains compromosing all logical mutations in all combinations of the genes that encode for NusA, NusG, and Rho, where we developed the first transcription termination atlas. We used this atlas to find that Rho can stimulate intrinsic termination in B. subtilis and we recapitulated this finding in vitro to dissect the mechanism.
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Project description:RNA polymerase II (RNAPII) transcription involves initiation from promoters, transcript elongation through the gene body, and cessation of transcription in the downstream terminator regions. In contrast to bacteria, where terminators often contain specific DNA elements to direct RNAP dissociation1, termination by RNAPII is thought to be driven entirely by protein co-factors1-3. Here we use biochemical reconstitution to shed new light on RNAPII termination. Unexpectedly, transcription through a terminator region by pure RNAPII results in a significant amount of intrinsic polymerase dissociation at specific sequences containing T-tracts. A combination of biochemistry and single molecule analysis indicates that such intrinsic termination involves pausing without backtracking prior to spontaneous RNAPII dissociation from the DNA template. Importantly, while the ‘torpedo’ Rat1-Rai1 RNA exonuclease (XRN2 in humans) works inefficiently on paused or stopped polymerases, it greatly stimulates intrinsic termination. By contrast, elongation factor Spt4-Spt5 (DSIF in humans) suppresses such termination. Genome-wide analysis in yeast using 3’-end sequencing further supports the idea that transcriptional termination occurs by transcript cleavage at the polyA site exposing a new RNA-end that allows loading of the Rat1-Rai1 torpedo, which then catches up with a destabilised RNAPII at intrinsic termination sites containing T-tracts to terminate transcription.
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Project description:RNA polymerase II (RNAPII) transcription involves initiation from promoters, transcript elongation through the gene body, and cessation of transcription in the downstream terminator regions. In contrast to bacteria, where terminators often contain specific DNA elements to direct RNAP dissociation1, termination by RNAPII is thought to be driven entirely by protein co-factors1-3. Here we use biochemical reconstitution to shed new light on RNAPII termination. Unexpectedly, transcription through a terminator region by pure RNAPII results in a significant amount of intrinsic polymerase dissociation at specific sequences containing T-tracts. A combination of biochemistry and single molecule analysis indicates that such intrinsic termination involves pausing without backtracking prior to spontaneous RNAPII dissociation from the DNA template. Importantly, while the ‘torpedo’ Rat1-Rai1 RNA exonuclease (XRN2 in humans) works inefficiently on paused or stopped polymerases, it greatly stimulates intrinsic termination. By contrast, elongation factor Spt4-Spt5 (DSIF in humans) suppresses such termination. Genome-wide analysis in yeast using 3’-end sequencing further supports the idea that transcriptional termination occurs by transcript cleavage at the polyA site exposing a new RNA-end that allows loading of the Rat1-Rai1 torpedo, which then catches up with a destabilised RNAPII at intrinsic termination sites containing T-tracts to terminate transcription.
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Project description:A unified allosteric/torpedo mechanism for transcriptional termination on human protein-coding genes
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Project description:Rho stimulates both Rho-dependent and intrinsic termination in Bacillus subtilis
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Project description:In this work we conducted Term-seq on Wild Type, NusA depletion, nusG deletion, and NusA depletion nusG deletion strains of B. subtilis. Using this approach, we found that NusG is an intrinsic termination factor that works single and cooperatively with NusA.
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