Core Mediator structure at 3.4 A extends transcription initiation complex model
Ontology highlight
ABSTRACT:
ORGANISM(S): Saccharomyces cerevisiae
PROVIDER: GSE95483 | GEO | 2017/03/01
SECONDARY ACCESSION(S): PRJNA377258
REPOSITORIES: GEO
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Project description:This study has determined structure of transcription initiation complexes including a DNA-bound activator, RNA polymerase II (Pol II), and Mediator on a divergent promoter GAL80/SUT719 using a combination of cryo-EM and XL-MS analyses. Our cryo-EM single-particle analysis reveals a dimeric form of Med-PIC through the Mediator Tail module induced by the activator protein. Density of the upstream DNA bound to the Gal4-VP16 was identifiable along the Mediator Tail module, while XL-MS localized flexible regions that were not visible by cryo-EM analysis, such as activator-binding domains (ABDs and KIX).
2024-05-22 | PXD036149 | Pride
Project description:Mediator is an essential, broadly utilized eukaryotic transcriptional co-activator. How and what it communicates from activators to RNA polymerase II remains an open question. Here we performed genome-wide location profiling of yeast Mediator subunits. Mediator is not found at core promoters but rather occupies the upstream activating sequence (UAS), upstream of the pre-initiation complex. In the absence of Kin28/CDK7 kinase activity, or in cells where the CTD is mutated to replace Ser5 with alanines, however, Mediator accumulates at core promoters together with RNAPII. We propose that Mediator is quickly released from promoters upon Ser5 phosphorylation by Kin28/CDK7, which also allows for RNAPII to escape from the promoter. We took a systematic approach to examine the genome-wide distribution (using ChIP-chip) of the various Mediator subunits. Mediator occupancy was also assayed in mutants for most of the CTD kinases and in strains where some CTD serines had been replaced by alanines. Mediator ChIPs were performed with Myc-tagged subunits, except in some preliminary experiments where polyclonal antibodies were used. Most ChIPs (in Cy5) were hybridyzed against a control ChIP sample from an isogenic non-tagged strain (in Cy3). In some of the preliminary experiments, non immunoprecipitated DNA (input) was used as the control. In addition to Mediator ChIPs, the project includes TFIIB and RNAPII (Rpb3) ChIP-chip datasets. All ChIP-chip experiments were done in duplicates except for the preliminary experiments that were done in monoplicat for the most part. Each microarray was normalized using the Lima Loess and replicates were combined using a weighted average method as previously described (Pokholok et al., 2005).
2014-03-24 | E-GEOD-55402 | biostudies-arrayexpress
Project description:MedPIC: Crosslinking-MS of 54 subunit pre-initiation complex from yeast.
MedPolII: Crosslinking-MS of 33 subunit Mediator + polII complex from yeast.
2016-08-02 | MSV000080013 | MassIVE
Project description:To test the role of Mpe1 in transcription termination we inserted a mini-auxin induced degron (mAID) at the C-terminal end of the endogenous MPE1 locus in the yeast Saccharomyces cerevisiae. We treated Mpe1-mAID cells (JRY101) or wild type cells (WT, YMK728) with 1 mM auxin for 30 minutes and labeled the nascent RNA with 4-thiouracil (4tU) for 6 minutes. The nascent RNA fraction was prepared by biotinylating the 4tU-labeled RNA followed purification with streptavidin beads. Nascent and total RNA fractions were depleted of ribosomal RNA and strand-specific libraries prepared. Libraries were single-end sequenced using an Ilummina HiSeq 4000 instrument.
2021-12-31 | E-MTAB-10820 | biostudies-arrayexpress
Project description:This project aims to understand the function of different mediator subunits (Med14 and Med17) in transcription in S. cerevisiae. Thus we analyzed total and newly synthesized RNA levels in conditional Med14 and Med17 depletion strains. For this purpose we used an auxin inducible degron system system to specifically induce the degradation Mediator subunits by addition of auxin into the culture medium. We measured transcript levels after 30 min auxin or DMSO (control) treatment induction of degradation of Med14 or Med17 by the addition of auxin (30 min) or with the carrier DMSO (control) followed by a 6 min labelling of newly synthesized RNA with 4-thiouracil (4tU). In parallel wiltype S.pombe cells were labelled with 4tU for 6min during midlog growth phase. Prior to RNA extraction (Ribopure Yeast Kit, Ambion) our labelled S.cerevisiae cells of interest were mixed with labelled S.pombe cells as a spike to allow normalization against an exogenous reference (ratio 3 S .cerevisiae for 1 S.pombe cell. From the total RNA we purified the 4tU labelled fraction of RNAs containing 4tU. Both the total RNA and the purified newly synthesized RNAs will then be subject to RNA-sequencing after ribosomal RNA depletion. In summary, we will provide 16 samples for this project containing RNA from both S.cerevisiae and S.pombe. The sample read counts were normalized with size factors computed by the median-of-ratios method proposed by Anders and Huber (Anders et al. 2010) on S. pombe genes to make these counts comparable between samples.
2020-03-04 | GSE146300 | GEO
Project description:We have combined biochemical purification of Mediator from chromatin with ChIP-sequencing to reveal Mediator occupupancy to DNA globally and to identify proteins interacting specifically with Mediator in chromatin. We find that Mediator occupy strong chromosomally interacting domain (CID) boundaries and nearly all tRNA genes. Purification of Mediator from chromatin shows that it interacts with proteins and protein complexes that have been shown to interact with CID boundaries such as RSC, Ssu72 and histone H4. We also show specific interaction between Mediator and the Arp2/Arp3, CPF, CF 1A and LSm, complexes in chromatin. These factors are involved in mRNA 3'-end processing, gene looping, actin assembly and mRNA decay.
2017-05-23 | GSE95051 | GEO
Project description:Mediator is an essential, broadly utilized eukaryotic transcriptional co-activator. How and what it communicates from activators to RNA polymerase II remains an open question. Here we performed genome-wide location profiling of yeast Mediator subunits. Mediator is not found at core promoters but rather occupies the upstream activating sequence (UAS), upstream of the pre-initiation complex. In the absence of Kin28/CDK7 kinase activity, or in cells where the CTD is mutated to replace Ser5 with alanines, however, Mediator accumulates at core promoters together with RNAPII. We propose that Mediator is quickly released from promoters upon Ser5 phosphorylation by Kin28/CDK7, which also allows for RNAPII to escape from the promoter.
2014-03-24 | GSE55402 | GEO
Project description:The Mediator complex plays an essential and multi-faceted role in regulation of RNA polymerase II transcription in all eukaryotes. Structural analysis of yeast Mediator provided an understanding of the conserved core of the complex and its interaction with RNA polymerase II, but failed to reveal how Mediator might enable a response to interaction with transcription factors (TFs). Here we present an atomic model of mammalian (Mus musculus) Mediator, derived from a 4.0 Å resolution cryo-EM map of the complex. The mammalian Mediator structure reveals how the previously unresolved Tail module, which includes a number of metazoan specific subunits, modulates Mediator conformation and interactions. The structure also suggests how a bi-partite organization of TF-interacting subunits can be exploited to enable a Mediator mechanism that combines conformational control of molecular interactions and enhancement of transcription through phase separation and enhancer-specific recruitment
2020-03-30 | GSE145902 | GEO
Project description:Mediator is a highly conserved transcriptional coactivator organized into four modules, namely Tail, Middle, Head and Kinase (CKM). Previous work suggests regulatory roles for Tail and CKM, but an integrated model for these activities is lacking. Here, we analyzed the genome-wide distribution of Mediator subunits in wild-type and mutant yeast cells in which RNA polymerase II promoter escape is blocked allowing detection of transient Mediator forms. We found that while all modules are recruited to upstream activated regions (UAS), assembly of Mediator within the pre-initiation complex is accompanied by the release of CKM. Interestingly, our data show that CKM regulates Mediator-UAS interaction rather than Mediator-promoter association. In addition, while Tail is required for Mediator recruitment to UAS, Tail-less Mediator nevertheless interacts with core promoters. Collectively, our data suggest that the essential function of Mediator is mediated by Head and Middle at core promoters, while Tail and CKM play regulatory roles.
2016-10-25 | GSE81107 | GEO
Project description:Mediator complex is an integrative hub for transcriptional regulation. Here we show that Mediator regulates alternative mRNA processing via its Med23 subunit. Combining tandem affinity purification and mass spectrometry, we identified a number of mRNA processing factors that bind to a soluble recombinant Mediator subunit MED23 but not to several other Mediator components. One of these factors, hnRNP L, specifically interacts with MED23 in vitro and in vivo. Consistently, Mediator partially colocalizes with hnRNP L and the splicing machinery in the cell. Functionally Med23 regulates a subset of hnRNP L-targeted alternative splicing (AS) and alternative cleavage and polyadenylation (APA) events as shown by minigene reporters and exon array analysis. ChIP-seq analysis revealed that Med23 can regulate hnRNP L occupancy at their co-regulated genes. Taken together, these results demonstrate a crosstalk between Mediator and the splicing machinery, suggesting a novel mechanism for coupling mRNA processing to transcription. Examination of hnRNP L and H3K36me3 enrichment in sictrl and si23 Hela cells
2012-02-24 | E-GEOD-33887 | biostudies-arrayexpress