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Functional studies of the yeast med5, med15 and med16 mediator tail subunits
Miriam Larsson1, Hanna Uvell, Jenny Sandström
1Department of Medical Biochemistry and Biophysics, Umeå University, Umeå, Sweden.
Abstract:
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. We identify 25 genes in each double mutant that show a significant change in expression when compared to the corresponding single mutants and to the wild type strain. Importantly, 13 of the 25 identified genes are common for both double mutants. We also find that all strains in which MED15 is inactivated show down-regulation of genes that have been identified as targets for the Ace2 transcriptional activator protein, which is important for progression through the G1 phase of the cell cycle. Supporting this observation, we demonstrate that loss of Med15 leads to a G1 arrest phenotype. Collectively, these findings provide insight into the function of the Mediator Tail module.
Insights
Investigating the yeast Mediator complex
Area of Science:
- Molecular Biology
- Yeast Genetics
- Gene Regulation
Background:
- The yeast Mediator complex is crucial for gene transcription, composed of Head, Middle, and Tail modules.
- The Tail module, containing Med2, Med3, Med5, Med15, and Med16, performs essential functions, even when detached from other modules.
- Synthetic lethality in MED5/MED15 and MED15/MED16 double mutants highlights the Tail module's critical role.
Purpose of the Study:
- To investigate the immediate effects of inactivating individual Mediator Tail subunits (Med5, Med15, Med16) on global gene expression.
- To analyze gene expression changes in double mutants (Med5/15 and Med15/16) compared to single mutants and wild-type strains.
- To elucidate the functional significance of the Mediator Tail module in yeast.
Main Methods:
- Utilized the N-Degron system to generate temperature-sensitive (ts) mutants for Mediator Tail subunits.
- Performed global gene expression analysis on single and double mutants.
- Investigated the impact of Med15 inactivation on Ace2 transcriptional targets and cell cycle progression.
Main Results:
- Identified 25 significantly altered genes in each double mutant (Med5/15 and Med15/16) compared to controls.
- Found 13 common genes with altered expression in both double mutants.
- Observed down-regulation of Ace2 target genes in all MED15-inactivated strains, leading to a G1 cell cycle arrest.
Conclusions:
- The Mediator Tail module plays a vital role in regulating specific gene sets, including Ace2 targets.
- Med15 is essential for proper cell cycle progression, specifically G1 phase.
- This study provides new insights into the functional organization and essential roles of the Mediator Tail module.
