| 1. |
- Balciunas, Darius, et al.
(författare)
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Functional interactions within yeast mediator and evidence of differential subunit modifications
- 2003
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Ingår i: Journal of Biological Chemistry. - 0021-9258 .- 1083-351X. ; 278:6, s. 3831-3839
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Tidskriftsartikel (refereegranskat)abstract
- It is possible to recruit RNA polymerase II to a target promoter and, thus, activate transcription by fusing Mediator subunits to a DNA binding domain. To investigate functional interactions within Mediator, we have tested such fusions of the lexA DNA binding domain to Med1, Med2, Gal11, Srb7, and Srb10 in wild type, med1, med2, gal11, sin4, srb8, srb10, and srb11 strains. We found that lexA-Med2 and lexA-Gal11 are strong activators that are independent of all Mediator subunits tested. lexA-Srb10 is a weak activator that depends on Srb8 and Srb11. lexA-Med1 and lexA-Srb7 are both cryptic activators that become active in the absence of Srb8, Srb10, Srb11, or Sin4. An unexpected finding was that lexA-VP16 differs from Gal4-VP16 in that it is independent of the activator binding Mediator module. Both lexA-Med1 and lexA-Srb7 are stably associated with Med4 and Med8, which suggests that they are incorporated into Mediator. Med4 and Med8 exist in two mobility forms that differ in their association with lexA-Med1 and lexA-Srb7. Within purified Mediator, Med4 is present as a phosphorylated lower mobility form. Taken together, these results suggest that assembly of Mediator is a multistep process that involves conversion of both Med4 and Med8 to their low mobility forms.
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| 2. |
- Balciunas, Darius
(författare)
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Functional studies in yeast of cyclin C and the RNA polymerase II Mediator complex
- 1999
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Cyclin C belongs to a group of cyclins that are not cell cycle-regulated. It was first cloned from Drosophila and rat, but its role was not understood until the yeast cyclin C homologue Srb 11 was identified in several genetic screens for transcriptional repressors and subsequently was shown to be associated with the RNA polymerase II Mediator complex. The Mediator is a multisubunit complex that enables RNA polymerase II to respond to activators in vitro.In the work presented here, the yeast genes encoding cyclin C (Srb11/Gig3), its cyclin-dependent kinase (Srb10/Gig2), and a third associated protein (Srb8/Gig1) were identified in a genetic screen for negative regulators of the gluconeogenic genes. A further analysis of the cloned genes suggested that the encoded proteins function closely together.The Med1 subunit of the yeast Mediator complex was characterized. Evidence was found of a functional connection between Med1 and the cyclin C-dependent kinase. The expression of the GAL1 promoter is partly deregulated in cells lacking cyclin C, Med1, or another mediator subunit, Med2. This deregulated expression is seen also under derepressed non-inducing conditions, and is therefore not due to a failure of glucose repression.An analysis of the ability of different Mediator subunits to activate transcription when fused to a DNA binding domain indicated that Med1 and Srb7 are negatively regulated both by cyclin C and by the Sin4 subunit of the Mediator, but not by the Med2 or Gal11 subunits, even though Sin4, Med2 and Gal11 are a part of the same module within the Mediator.A screen was made for multicopy suppressors of disruptions in the SRB8, SRB10 and SRB11 genes. Since these disruptions lack selectable phenotypes in a wild type background, the failure of snf1 mig1 srb8/10/11 cells to grow on galactose was used to select suppressors. Four new genes were identified and named GISI-4. Evidence was obtained of a functional interaction between these genes and the RAS/cAMP pathway.
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| 3. |
- Balciunas, Darius, et al.
(författare)
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The Med1 subunit of the yeast mediator complex is involved in both transcriptional activation and repression
- 1999
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Ingår i: Proceedings of the National Academy of Sciences of the United States of America. - : Proceedings of the National Academy of Sciences (PNAS). - 0027-8424 .- 1091-6490. ; 96:2, s. 376-381
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Tidskriftsartikel (refereegranskat)abstract
- The mediator complex is essential for regulated transcription in vitro. In the yeast Saccharomyces cerevisiae, mediator comprises >15 subunits and interacts with the C-terminal domain of the largest subunit of RNA polymerase II, thus forming an RNA polymerase II holoenzyme. Here we describe the molecular cloning of the MED1 cDNA encoding the 70-kDa subunit of the mediator complex. Yeast cells lacking the MED1 gene are viable but show a complex phenotype including partial defects in both repression and induction of the GAL genes. Together with results on other mediator subunits, this implies that the mediator is involved in both transcriptional activation and repression. Similar to mutations in the SRB10 and SRB11 genes encoding cyclin C and the cyclin C-dependent kinase, a disruption of the MED1 gene can partially suppress loss of the Snf1 protein kinase. We further found that a lexA-Med1 fusion protein is a strong activator in srb11 cells, which suggests a functional link between Med1 and the Srb10/11 complex. Finally, we show that the Med2 protein is lost from the mediator on purification from Med1-deficient cells, indicating a physical interaction between Med1 and Med2.
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| 4. |
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| 5. |
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| 6. |
- Hallberg, Magnus, et al.
(författare)
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Functional and physical interactions within the middle domain of the yeast mediator
- 2006
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Ingår i: Molecular Genetics and Genomics. - : Springer Science and Business Media LLC. - 1617-4615 .- 1617-4623. ; 276:2, s. 197-210
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Tidskriftsartikel (refereegranskat)abstract
- Med21 (Srb7) is a small essential subunit of the middle domain of the Mediator, which is conserved in all eukaryotes. It is thought to play an important role in both transcriptional activation and repression. In the yeast Saccharomyces cerevisiae, Med21 is known to interact both with the Mediator subunit Med6 and the global co-repressor Tup1. We have made a temperature-sensitive med21-ts mutant, which we used in a high copy number suppressor screen. We found ten yeast genes that can suppress the med21-ts mutation in high copy number. The three strongest suppressors were MED7 and MED10 (NUT2), which encode other Mediator subunits, and ASH1, which encodes a repressor of the HO gene. 2-Hybrid experiments confirmed multiple interactions between Med21, Med10, Med7 and Med4, and also revealed a Med21 self-interaction. The interactions of Med21 with Med7 and Med10 were verified by co-immunoprecipitation of tagged proteins produced in insect cells and E. coli, where both interactions were found to depend strongly on the amino acid residues 2-8 of Med21. These interactions, and the interactions of Med21 with Med6 and Tup1, suggest that Med21 may serve as a molecular switchboard that integrates different signals before they reach the core polymerase.
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| 7. |
- Tronnersjö, Susanna, et al.
(författare)
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The jmjN and jmjC domains of the yeast zinc finger protein Gis1 interact with 19 proteins involved in transcription, sumoylation and DNA repair
- 2007
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Ingår i: Molecular Genetics and Genomics. - : Springer Science and Business Media LLC. - 1617-4615 .- 1617-4623. ; 277:1, s. 57-70
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Tidskriftsartikel (refereegranskat)abstract
- The jumonji domain is a highly conserved bipartite domain made up of two subdomains, jmjN and jmjC, which is found in many eukaryotic transcription factors. The jmjC domain was recently shown to possess the histone demethylase activity. Here we show that the jmjN and jmjC domains of the yeast zinc finger protein Gis1 interact in a two-hybrid system with 19 yeast proteins that include the RecQ helicase Sgs1, the silencing factors Esc1 and Sir4, the URI-type prefoldin Bud27 and the PIAS type SUMO ligase Nfi1/Siz2. Extensive interaction cross dependencies further suggest that the proteins form a larger complex. Consistent with this, 16 of the proteins also interact with a Bud27 two-hybrid bait, and three of them co-precipitate with TAP-tagged Gis1. The Gis1 jumonji domain can repress transcription when recruited to a promoter as a lexA fusion. This effect is dependent on both the jmjN and jmjC subdomains, as were all 19 two-hybrid interactions, indicating that the two subdomains form a single functional unit. The human Sgs1 homolog WRN also interacts with the Gis1 jumonji domain. Finally, we note that several jumonji domain interactors are related to proteins that are found in mammalian PML nuclear bodies.
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