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Dynamics of protein folding and subunit interactions in assembly of the yeast mediator complex
Umeå University, Faculty of Medicine, Medical Biochemistry and Biophsyics. (Stefan Björklund)
2009 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The Mediator complex was originally discovered in the yeast Saccharomyces cerevisiae and has since then been shown to be required for transcriptional regulation both in vitro and in vivo. The Mediator complex also stimulates basal, unregulated transcription and serves as a bridge by conveying signals from promoter-bound transcriptional regulatory proteins such as activators and repressors to the RNA Polymerase II general transcriptional machinery. The Mediator consists of 21 subunits and can be divided into three distinct modules head, middle and tail.

Despite the tremendous progress that has been achieved so far in characterizing the Mediator complex both functionally and structurally, many aspects of the complex are not yet well understood. The objective of this work is to achieve further understanding of the Mediator complex by studying the folding of different protein subunits, their interactions and how that affects assembly of the Mediator complex.

In our first study we made a temperature-sensitive med21 mutant and used it to identify genes that can suppress the mutation when present in high copy number. Among the 10 genes that we identified, the strongest suppressors were Med7 and Med10, which encode Mediator subunits, and Ash1, which encodes a repressor of the HO gene. We also used 2-hybrid experiments and immunoprecipitation to study protein-protein interactions between Med21 and the Med4, Med7 and Med10 proteins which are all essential for viability and located within the middle domain of the Mediator complex. We found that the N-terminal 2-8 amino acids of Med21 are required for interactions with Med7 and Med10. These results led us to propose a model in which the N-terminal part of Med21 functions as a molecular switchboard where competing signals from various activators, repressors and mediator subunits are integrated prior to reaching the general transcription machinery.

In our second study, we extended our studies of protein-protein interactions to another part of the mediator complex by studying the folding and the assembly processes of the mediator head domain subunits Med8, Med18 and Med20. Using purified proteins and a combination of several different methods such as immunoprecipitation, far-UV circular dichroism and fluorescence, we demonstrated that the Med8, Med18 and Med20 subunits are interdependent on each other for proper folding and complex formation.

Place, publisher, year, edition, pages
Umeå: Umeå university , 2009. , 44 p.
Umeå University medical dissertations, ISSN 0346-6612 ; 1290
Keyword [en]
mediator, transcriptional regulation, assembly, folding
National Category
Medical and Health Sciences
Research subject
Medical Biochemistry
URN: urn:nbn:se:umu:diva-29976ISBN: 978-91-7264-854-8OAI: diva2:278788
Public defence
2010-01-12, KB3B1, KBC house, 901 87 Umeå, Umeå University, 10:00 (English)
Available from: 2009-12-21 Created: 2009-11-30 Last updated: 2009-12-21Bibliographically approved
List of papers
1. Functional and physical interactions within the middle domain of the yeast mediator
Open this publication in new window or tab >>Functional and physical interactions within the middle domain of the yeast mediator
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2006 (English)In: Molecular Genetics and Genomics, ISSN 1617-4615, Vol. 276, no 2, 197-210 p.Article in journal (Refereed) Published
Abstract [en]

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, Gene Expression Regulation; Fungal/*genetics, Molecular Sequence Data, Multiprotein Complexes/genetics/metabolism, Mutation, Nuclear Proteins/genetics/metabolism, Protein Binding/genetics, Saccharomyces cerevisiae/*genetics/metabolism, Saccharomyces cerevisiae Proteins/*genetics/metabolism, Signal Transduction, Trans-Activation (Genetics)/*genetics, Trans-Activators/genetics/metabolism, Transcription Factors/*genetics/metabolism, Two-Hybrid System Techniques
National Category
Cell and Molecular Biology
Research subject
Molecular Biology
urn:nbn:se:umu:diva-6334 (URN)10.1007/s00438-006-0135-7 (DOI)16758199 (PubMedID)
Available from: 2007-12-09 Created: 2007-12-09 Last updated: 2009-12-21Bibliographically approved
2. Med8, Med18, and Med20 subunits of the Mediator head domain are interdependent upon each other for folding and complex formation
Open this publication in new window or tab >>Med8, Med18, and Med20 subunits of the Mediator head domain are interdependent upon each other for folding and complex formation
2009 (English)In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 106, no 49, 20728-20733 p.Article in journal (Refereed) Published
Abstract [en]

We have studied folding and complex formation of the yeast mediator head-module protein subunits Med8, Med18, and Med20. Using a combination of immunoprecipitation, far-UV circular dichroism, and fluorescence measurements on recombinantly expressed and denatured proteins that were allowed to renature separately or in different combinations, we found that Med8, Med18, and Med20 can fold in different ways to form both soluble monomeric proteins and different distinct subcomplexes. However, the concurrent presence of all three protein subunits during the renaturation process is required for proper folding and trimer complex formation.

National Category
Biochemistry and Molecular Biology
Research subject
urn:nbn:se:umu:diva-29965 (URN)10.1073/pnas.0907645106 (DOI)
Available from: 2009-11-30 Created: 2009-11-30 Last updated: 2012-08-15Bibliographically approved

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