DNA-directed termination of RNA Polymerase II transcription
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PROVIDER: PRJNA906143 | ENA |
REPOSITORIES: ENA
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Project description:DNA-directed termination of RNA Polymerase II transcription II
| PRJNA906147 | ENA
Project description:DNA-directed termination of RNA Polymerase II transcription I
| PRJNA906145 | ENA
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.
2023-09-11 | GSE218927 | GEO
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.
2023-09-11 | GSE218924 | GEO
Project description:We report a function of human mRNA decapping factors in control of transcription by RNA polymerase II. Decapping proteins Edc3, Dcp1a and Dcp2 and the termination factor TTF2 co-immunoprecipitate with Xrn2, the nuclear 5'-3' exonuclease torpedo that facilitates transcription termination at the 3' ends of genes. Dcp1a, Xrn2 and TTF2 localize near transcription start sites (TSSs) by ChIP-Seq. At genes with 5' peaks of paused pol II, knockdown of decapping or termination factors, Xrn2 and TTF2, shifted polymerase away from the TSS toward upstream and downstream distal positions. This re-distribution of pol II is similar in magnitude to that caused by depletion of the elongation factor Spt5. We propose that coupled decapping of nascent transcripts and premature termination by the torpedo mechanism is a widespread mechanism that limits bidirectional pol II elongation. Regulated co-transcriptional decapping near promoter-proximal pause sites followed by premature termination could control productive pol II elongation.
2012-04-05 | GSE36185 | GEO
Project description:We report a function of human mRNA decapping factors in control of transcription by RNA polymerase II. Decapping proteins Edc3, Dcp1a and Dcp2 and the termination factor TTF2 co-immunoprecipitate with Xrn2, the nuclear 5'-3' exonuclease torpedo that facilitates transcription termination at the 3' ends of genes. Dcp1a, Xrn2 and TTF2 localize near transcription start sites (TSSs) by ChIP-Seq. At genes with 5' peaks of paused pol II, knockdown of decapping or termination factors, Xrn2 and TTF2, shifted polymerase away from the TSS toward upstream and downstream distal positions. This re-distribution of pol II is similar in magnitude to that caused by depletion of the elongation factor Spt5. We propose that coupled decapping of nascent transcripts and premature termination by the torpedo mechanism is a widespread mechanism that limits bidirectional pol II elongation. Regulated co-transcriptional decapping near promoter-proximal pause sites followed by premature termination could control productive pol II elongation. RNA pol II (GSE30895: GSM766171), Xrn2, TTF2 and Dcp1a were localized by ChIP-Seq in HeLa cells. RNA pol II was localized in control HEK293 cells and cells infected with lentiviruses expressing a scrambled control shRNA (scr), and shRNAs targeting the following proteins: Xrn2, TTF2, Xrn2+TTF2, Edc3, Dcp1a, and Dcp2.
2012-04-04 | E-GEOD-36185 | biostudies-arrayexpress
Project description:At the 3'-ends of genes, RNA polymerase (Pol) II is dephosphorylated at tyrosine 1 residues of its C-terminal domain (CTD), resulting in recruitment of transcription termination factors. We show that the multisubunit cleavage and polyadenylation factor (CPF) is a Pol II CTD phosphatase and its Glc7 subunit is required for Tyr1 dephosphorylation at the poly-adenylation site and Pol II termination in vivo. ChIP-chip was performed to examine the effect of Glc7 nuclear depletion on genome-wide Pol II occupancy [using ?-Rpb3 (1Y26, cat. no. W0012, neoclone) antibody] and CTD tyrosine 1 phosphorylation levels [using ?-TyrY1P (3D12, D. Eick) antibody].
2013-12-16 | E-MTAB-1528 | biostudies-arrayexpress
Project description:CDK9 is a kinase critical for the productive transcription of protein-coding genes by RNA polymerase II (pol II). As part of P-TEFb, CDK9 phosphorylates the carboxyl-terminal domain (CTD) of pol II and elongation factors, which allows pol II to elongate past the early elongation checkpoint (EEC) encountered soon after initiation. We show that, in addition to halting pol II at the EEC, loss of CDK9 activity causes premature termination of transcription across the last exon, loss of polyadenylation factors from chromatin, and loss of polyadenylation of nascent transcripts. Inhibition of the phosphatase PP2A abrogates the premature termination and loss of polyadenylation caused by CDK9 inhibition, indicating that this kinase/phosphatase pair regulates transcription elongation and RNA processing at the end of protein-coding genes. We also confirm the splicing factor SF3B1 as a target of CDK9 and show that SF3B1 in complex with polyadenylation factors is lost from chromatin after CDK9 inhibition. These results emphasize the important roles that CDK9 plays in coupling transcription elongation and termination to RNA maturation downstream of the EEC.
2022-08-22 | GSE176541 | GEO
Project description:The RNA Exosome Promotes Transcription Termination of backtracked RNA Polymerase II
| PRJEB5240 | ENA
Project description:RNA PolyRNA Polymerase II CTD phosphatase Rtr1 prevents premature transcription termination
| PRJNA557233 | ENA