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The Med1 subunit of the yeast mediator complex is involved in both transcriptional activation and repression
D Balciunas1, C Gälman, H Ronne
1Department of Medical Biochemistry and Microbiology, Uppsala University Biomedical Center, Box 582, 751 23 Uppsala, Sweden.
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.
Insights
The MED1 gene encodes a key mediator complex subunit. Its absence causes defects in gene regulation and suggests a link between Med1 and Srb10/11, impacting transcription.
Area of Science:
- Molecular Biology
- Gene Regulation
- Yeast Genetics
Background:
- The mediator complex is crucial for transcription regulation.
- It comprises over 15 subunits and interacts with RNA polymerase II.
- Understanding subunit functions is vital for elucidating transcriptional control.
Purpose of the Study:
- To molecularly clone the MED1 cDNA, encoding a 70-kDa mediator subunit.
- To investigate the role of MED1 in gene transcription in Saccharomyces cerevisiae.
- To explore functional links between MED1 and other regulatory proteins.
Main Methods:
- Molecular cloning of MED1 cDNA.
- Gene disruption in yeast to create MED1-deficient cells.
- Analysis of GAL gene expression and genetic interactions.
- Protein interaction studies using lexA-Med1 fusion and co-purification.
Main Results:
- MED1 gene disruption leads to partial defects in GAL gene repression and induction.
- MED1 disruption partially suppresses the loss of Snf1 protein kinase.
- A lexA-Med1 fusion protein acts as a strong activator in srb11 cells.
- Med2 protein is lost from the mediator complex in Med1-deficient cells, indicating physical interaction.
Conclusions:
- The mediator complex, including MED1, is involved in both transcriptional activation and repression.
- MED1 functionally interacts with the Srb10/11 complex and Snf1 kinase.
- MED1 physically interacts with the Med2 subunit, highlighting its role in mediator complex integrity and function.