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Stefan Björklund

Publications and source records attributed to Stefan Björklund.

7 recordsLinked to original sources
PubMed2006

Functional and physical interactions within the middle domain of the yeast mediator.

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.

Amino Acid Sequence↗
PubMed2005

The yeast Mediator complex and its regulation.

The Mediator complex acts as a bridge, conveying regulatory information from enhancers and other control elements to the basal RNA polymerase II transcription machinery. Mediator is required for the regulated transcription of nearly all RNA polymerase II-dependent genes in Saccharomyces cerevisiae, and post-translational modifications of specific Mediator subunits can affect global patterns of gene transcription.

Animals↗
PubMed2004

Site-specific Srb10-dependent phosphorylation of the yeast Mediator subunit Med2 regulates gene expression from the 2-microm plasmid.

The yeast Mediator complex is required for transcriptional regulation both in vivo and in vitro, and its function is conserved in all eukaryotes. Mediator interacts with both transcriptional activators and RNA polymerase II, but little is known about the mechanisms by which it operates at the molecular level. Here, we show that the cyclin-dependent kinase Srb10 interacts with, and phosphorylates, the Med2 subunit of Mediator both in vivo and in vitro. A point mutation of the single phosphorylation site in Med2 results in a strongly reduced expression of the REP1, REP2, FLP1, and RAF1 genes, which are all located on the endogenous 2-microm plasmid. Combined with previous studies on the effects of SRB10/SRB11 deletions, our data suggest that posttranslational modifications of Mediator subunits are important for regulation of gene expression.

Amino Acid Sequence↗
PubMed2003

S Phase-specific transcription of the mouse ribonucleotide reductase R2 gene requires both a proximal repressive E2F-binding site and an upstream promoter activating region.

Ribonucleotide reductase is essential for supplying a balanced pool of the four deoxyribonucleotides required for DNA synthesis and repair. The active enzyme consists of two non-identical subunits called proteins R1 and R2. There are multiple levels of regulation of ribonucleotide reductase activity, which is highest during the S and G(2) phases of an unperturbed cell cycle in mammalian cells. Previous reports in the literature have indicated that the S phase-specific transcription of the mammalian R2 gene is regulated by a transcriptional block, is dependent on the transcription factor E2F1, or is simply regulated by proteins that bind to promoter CCAAT boxes plus the TATA box. Here, we demonstrate that the S phase-specific transcription of the mouse R2 gene is dependent on an upstream promoter activating region (located at nucleotides (nt) -672 to -527 from the transcription start site) and one proximal promoter repressive element (located at nt -112 to -107) that binds E2F4. Binding to the E2F site is modulated by binding of nuclear factor-Y to an adjacent CCAAT element (nt -79 to -75). The upstream activating region is crucial for overall R2 promoter activity. Mutation of the E2F-binding site leads to premature promoter activation in G(1) and increases overall promoter activity but only when the upstream activating region is present and intact. Therefore, E2F-dependent repression is essential for cell cycle-specific R2 transcription.

Animals↗
PubMed2003

Sequences downstream of the transcription initiation site are important for proper initiation and regulation of mouse ribonucleotide reductase R2 gene transcription.

Ribonucleotide reductase is essential for the synthesis of all four dNTPs required for DNA replication. The enzyme is composed of two proteins, R1 and R2, which are both needed for activity. Expression of the R1 and R2 mRNAs is restricted to the S-phase of the cell cycle, but the R1 and R2 promoters show no obvious sequence homologies that could indicate coordination of transcription. Here we study initiation of transcription at the natural mouse R2 promoter, which contains an atypical TATA-box with the sequence TTTAAA, using a combination of in vivo reporter gene assays and in vitro transcription. Our results indicate that in constructs where sequences from the R2 5'-UTR are present, the mouse R2 TATA-box is dispensable both for unregulated, basal transcription from the R2 promoter and for S-phase specific activity. Instead, initiation of R2 transcription is directed by sequences downstream from the transcription start. We report that this region contains a conserved palindrome sequence that interacts with TAFIIs. This interaction down-regulates basal transcription from the R2 promoter, both in the absence and in the presence of the TATA-box.

5' Untranslated Regions↗
PubMed2002

Functional interactions within yeast mediator and evidence of differential subunit modifications.

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.

Base Sequence↗