Saccharomyces cerevisiae
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ABSTRACT:
PROVIDER: PRJNA207349 | ENA |
REPOSITORIES: ENA
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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:Effect of loss of function of Gal11/Med15 and Med3 from the Mediator tail module in budding yeast
| PRJNA145277 | ENA
Project description:Our mechanistic understanding of Mediator derives largely from studies of the 25-subunit yeast complex. Here we combine CRISPR-Cas9 genetic screens, degron assays, in situ Hi-C, and cryo-EM to dissect the function and structure of the 33-subunit mammalian Mediator (mMED). Deletion analyses in B, T and ES cells reveal that depletion of the entire complex blocks PolII recruitment genome-wide, while loss of non-essential subunits, including the Tail module, primarily affects promoters linked to multiple enhancers. Contrary to current models, we find that mMED is not required to tether regulatory DNA, a topological activity controlled predominantly by architectural proteins. Structurally, we show that alterations in the Tail module, particularly at the core-Tail interphase, effect crucial mMED-PolII contacts, providing a rationale for how TFs stabilize the mMED-PolII holoenzyme and promote gene expression. Our studies therefore reveal key insights into how Mediator functionally bridge promoters and enhancers to regulate transcription initiation in higher eukaryotes.
2019-08-01 | GSE121355 | GEO
Project description:Mediator is a multiprotein transcriptional co-regulator complex composed of four modules; Head, Middle, Tail, and Kinase. It conveys signals from promoter-bound transcriptional regulators to RNA polymerase II and thus plays an essential role in eukaryotic gene regulation. We describe subunit localization and activities of Mediator in Arabidopsis through metabolome and transcriptome analyses from a set of Mediator mutants. Functional metabolomic analysis based on the metabolite profiles of Mediator mutants using multivariate statistical analysis and heat-map visualization shows that different subunit mutants display distinct metabolite profiles, which cluster according to the reported localization of the corresponding subunits in yeast. Based on these results, we suggest localization of previously unassigned plant Mediator subunits to specific modules. We also describe novel roles for individual subunits in development, and demonstrate changes in gene expression patterns and specific metabolite levels in med18 and med25, which can explain their phenotypes. We find that med18 displays levels of phytoalexins normally found in wild type plants only after exposure to pathogens. Our results indicate that different Mediator subunits are involved in specific signaling pathways that control developmental processes and tolerance to pathogen infections.
2018-01-30 | MTBLS450 | MetaboLights
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. Surprisingly, 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.
2016-10-20 | GSE81127 | 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:Transcriptional repression of ribosomal components and tRNAs is coordinately regulated in response to a wide variety of environmental stresses. Part of this response involves the convergence of different nutritional and stress signaling pathways on Maf1, a protein that is essential for repressing transcription by RNA polymerase (pol) III in Saccharomyces cerevisiae. Here we identify the functions buffering yeast cells that are unable to down-regulate transcription by RNA pol III. MAF1 genetic interactions identified in screens of non-essential gene-deletions and conditionally-expressed essential genes reveal a highly interconnected network of 64 genes involved in ribosome biogenesis, RNA pol II transcription, tRNA modification, ubiquitin-dependent proteolysis and other processes. A survey of non-essential MAF1 synthetic sick/lethal (SSL) genes identified six gene-deletions that are defective in transcriptional repression of ribosomal protein (RP) genes following rapamycin treatment. This subset of MAF1 SSL genes included MED20 which encodes a head module subunit of the RNA pol II Mediator complex. Genetic interactions between MAF1 and subunits in each structural module of Mediator were investigated to examine the functional relationship between these transcriptional regulators. Gene expression profiling identified a prominent and highly selective role for Med20 in the repression of RP gene transcription following treatments with rapamycin, chlorpromazine and tunicamycin and in post-diauxic cells. In addition, attenuated repression of RP genes by rapamycin was observed in a strain deleted for the Mediator tail module subunit Med16. The data suggest that Mediator and Maf1 function in parallel pathways to negatively regulate RP mRNA and tRNA synthesis. Keywords: genetic modification, stress response We generated 12 microarray profiles from fluor-reversed replicates of wild-type and med20? strains with or without treatment with rapamycin and under 5 other conditions that repress ribosomal protein gene transcription. The effect of rapamycin on strains deleted for 3 other Mediator subunits was also assessed relative to wild-type. See Willis et al., (2008) in revision.
2008-06-01 | E-GEOD-11397 | biostudies-arrayexpress
Project description:Eukaryotic RNA polymerase II (RNAPII) transcribes mRNA genes and non-protein coding RNAs (ncRNAs) including small nuclear and nucleolar RNAs (sn/snoRNAs). In metazoans, RNAPII transcription of sn/snoRNAs is facilitated by a number of specialized complexes, but no such complexes have been discovered in yeast. It has been proposed that yeast sn/snoRNA promoters use the same factors as mRNA promoters, but the extent to which regulators of mRNA genes function at yeast sn/snoRNA genes is unclear. Here, we investigated a potential role for the Mediator complex, essential for mRNA gene transcription, in the transcription of sn/snoRNA genes. We found that the Mediator maps to most sn/snoRNA gene regulatory regions and that rapid depletion of the essential structural subunit Med14 strongly reduces RNAPII and TFIIB occupancy as well as nascent transcription of sn/snoRNA genes. Deletion of Med3 and Med15, subunits of the activator-interacting Mediator tail module, does not affect Mediator recruitment to or RNAPII and TFIIB occupancy of sn/snoRNA genes. Our analyses suggest that Mediator promotes PIC formation and transcription at sn/snoRNA genes, expanding the role of this critical regulator beyond its known functions in mRNA gene transcription and demonstrating further mechanistic similarity between the transcription of mRNA and sn/snoRNA genes.
2018-10-08 | GSE112721 | GEO
Project description:The yeast Mediator complex can be divided into three modules, designated Head, Middle and Tail. Tail comprises the Med2, Med3, Med5, Med15 and Med16 protein subunits, which are all encoded by genes that are individually non-essential for viability. In cells lacking Med16, Tail is displaced from Head and Middle. However, inactivation of MED5/MED15 and MED15/MED16 are synthetically lethal, indicating that Tail performs essential functions as a separate complex even when it is not bound to Middle and Head. We have used the N-Degron method to create temperature sensitive (ts) mutants in the Mediator tail subunits Med5, Med15 and Med16 to study the immediate effects on global gene expression when each subunit is individually inactivated, and when MED5/15 or MED15/16 are inactivated together.
2013-06-07 | GSE47712 | GEO