Forschungsarbeit
A structure-function analysis of the transcription coactivator complex Mediator
By Martin Seizl (29.08.2011)
Transcription is the fundamental biological process of synthesizing RNA from a DNA template by RNA polymerase. In eukaryotes regulation of RNA polymerase (Pol) II transcription represents the basis for fundamental biological phenomena, including cell differentiation, organism development and biodiversity. The regulation of transcription initiation is the crucial step involving hundreds of activator and repressor proteins (1). During activation of a gene the so-called preinitiation complex (PIC), comprising Pol II, the general transcription factors (GTF) and coactivator complexes, has to assemble at the promoter (Figure 1). Coactivators are necessary to integrate multiple regulatory signals and transmit an integrated output to the basal transcription machinery. Together with the SAGA complex, the multiprotein complex Mediator is the most prominent coactivator (2). Mediator is required for regulated transcription of most if not all protein-coding genes and is conserved throughout eukaryotes. Recent studies have linked mutations in Mediator with various human diseases, including Alzheimer disease, congenital malformations, mental retardation and cancer. The Mediator complex from the yeast Saccharomyces cerevisiae consists of 25 subunits all having homologs in higher eukaryotes. It comprises a total molecular weight of around 1.5 MDa. Based on observations from electron microscopy and biochemical studies the Mediator complex can be subdivided into four modules referred to as head, middle, tail and the dissociable kinase module. Despite Mediator’s fundamental role in gene regulation, the molecular mechanisms underlying its function are still poorly understood.
[Bildunterschrift / Subline]: Figure 1: Schematic view of the Pol II preinitiation complex at the core promoter. Mediator bridges between activators (Act) bound to regulatory DNA elements (RE) and the basal transcription machinery (Pol II and the GTF). Pol II subsequently starts synthesizing RNA at the so-called Initiator DNA element (INR). Mediator modules are colored in blue (head), green (middle), magenta (tail) and orange (kinase). Individual Mediator subunits are shown. During my PhD project I was working towards a detailed structure-function analysis of the yeast Mediator complex. I focused on the highly conserved 7-subunit head module, which contacts Pol II and several general transcription factors. I used a combination of X-ray crystallography, yeast genetics, biochemical assays, chromatin immunoprecipitation and genome-wide gene expression profiling to elucidate the submodular architecture and molecular basis underlying Mediator function. The conserved functional head submodules Med8C/18/20 (3) and Med11/22 (4) were identified and their distinct functions characterized. Conserved flexible anchoring modes to the head module were demonstrated for both submodules. Structure-guided mutagenesis of Med11/22 identified a highly conserved surface patch (Figure 2A & 2B) required for stable preinitiation complex formation in vitro and in vivo. While the non-essential Med8C/18/20 submodule is required for low transcription levels of a specific subset of nonactivated genes, mutations in the essential Med11/22 submodule had a pleiotropic effect on gene expression (Figure 2C). Furthermore, the determined crystal structure of Med11/22 revealed an unexpected homology to the Med7/21 middle module subcomplex (Figure 2D). Structure predictions identified a total of 9 out of 17 Mediator core subunits and two metazoan-specific subunits sharing the heterodimeric four-helix bundle fold of Med11/22. During evolution this common structural building block appears to have duplicated and diversified to generate new protein interaction surfaces and thus accommodate the need for more complex regulatory mechanisms.
[Bildunterschrift / Subline]: Figure 2: Structural and functional characterization of Mediator head submodules. (A) Crystal structure of Saccharomyces cerevisiae Mediator subunits Med11 and Med22, colored in brown and cyan, respectively. (B) Surface conservation from yeast to human of the Med11/22 heterodimer. Invariant and conserved residues are shown in green and yellow, respectively. The conserved surface patch is marked by a dashed line. (C) Genome-wide gene expression profiling of Med11/22 mutant yeast strains. For comparison profiles of deletion mutants, namely med7N/31Δ (middle module), med20Δ (Med8C/18/20 submodule) and med2Δ (tail module) are shown. Horizontal lines represent individual yeast genes (yellow = up-regulated, black = no change, blue = down-regulated). (D) Structural similarity of Med11/22 (brown/cyan) and Med7/21 (orange/magenta). Taken together, my results contribute to the understanding of the architecture, function and evolution of the central transcriptional coactivator Mediator. Furthermore, the crystal structure of Med11/22 puts previously described mutations into a molecular context and will serve as the basis for future functional studies.
References:
1. Sikorski,T.W. and Buratowski,S. (2009) The basal initiation machinery: beyond the general transcription factors. Curr Opin Cell Biol, 21, 344-351, 10.1016/j.ceb.2009.03.006.
2. Malik,S. and Roeder,R.G. (2010) The metazoan Mediator co-activator complex as an integrative hub for transcriptional regulation. Nat Rev Genet, 11, 761-772, 10.1038/nrg2901.
3. Larivière,L., Seizl,M., van Wageningen,S., Röther,S., van de Pasch,L., Feldmann,H., Strässer,K., Hahn,S., Holstege,F.C.P. and Cramer,P. (2008) Structure-system correlation identifies a gene regulatory Mediator submodule. Genes Dev, 22, 872-877, 10.1101/gad.465108.
4. Seizl,M., Larivière,L., Pfaffeneder,T., Wenzeck,L. and Cramer,P. (2011) Mediator head subcomplex Med11/22 contains a common helix bundle building block with a specific function in transcription initiation complex stabilization. Nucleic Acids Res, 10.1093/nar/gkr229.
- Since 05/11
- PostDoc: University of Munich (LMU) – Gene Center, Munich, Germany, Prof. Patrick Cramer
- 05/07 – 04/11
- PhD thesis: University of Munich (LMU) – Gene Center, Munich, Germany, Prof. Patrick Cramer “The Mediator head module and regulation of RNA polymerase II transcription initiation”
- 11/06 – 04/07
- Master’s thesis: Fred Hutchinson Cancer Research Center, Seattle, USA; Gene Center, University of Munich (LMU), Munich, Germany, Prof. Steve Hahn & Prof. Patrick Cramer “A structure-function analysis of the transcription coactivator complex Mediator”
- 10/05 – 04/07
- Biochemistry (M.Sc.): University of Munich (LMU), Munich, Germany
- 04/05 – 09/05
- Industry research internship: Crelux GmbH, Martinsried, Germany
- 04/03 – 07/03
- Bachelor’s thesis: Institute of Microbiology, Technical University of Munich (TUM), Weihenstephan, Germany - Prof. Siegfried Scherer “Degradation of PrP-res by proteases of coryneform bacteria”
- 10/01 – 03/05
- Human nutrition (B.Sc.): Technical University of Munich (TUM), Weihenstephan, Germany; Oklahoma State University, Stillwater, USA
- 07/00 – 04/01
- German military service
- 09/91 – 06/00
- Abitur: Gymnasium (Secondary School), Kirchheim, Germany
- Seizl M, Larivière L, Pfaffeneder T, Wenzeck L, Cramer P. A conserved four-helix bundle in Mediator subunits Med11/Med22 is required for stable preinitiation complex formation. Nucleic Acids Res. Epub 2011 Apr 15.
- Chanarat S, Seizl M, Straesser K. The Prp19 complex is a novel transcription elongation factor. Genes Dev. 2011 in press.
- Czeko E, Seizl M, Mielke T, Cramer P. Iwr1 directs RNA polymerase II nuclear import. Mol. Cell. 2011 in press.
- Vojnić E, Mourão A, Seizl M, Simon B, Wenzeck L, Larivière L, Baumli S, Baumgart K, Meisterernst M, Sattler M, Cramer P. The Mediator Med25 activator interaction domain: Structure and cooperative binding of VP16 subdomains. Nat Struct Mol Biol. 2011 Apr;18
- Kostrewa D, Zeller ME, Armache KJ, Seizl M, Leike K, Thomm M, Cramer P. RNA polymerase II-TFIIB structure and mechanism of transcription initiation. Nature. 2009 Nov 19;462(7271):323-30
- Koschubs T, Seizl M, Larivière L, Kurth F, Baumli S, Martin DE, Cramer P. Identification, structure, and functional requirement of the Mediator submodule Med7N/31. EMBO J. 2009 Jan 7;28(1):69-80. Epub 2008 Dec 4
- Lariviere L, Seizl M, van Wageningen S, Roether S, Feldmann H, Straesser K, Hahn S, Holstege F, Cramer P. Structure-system correlation identifies a gene regulatory Mediator submodule. Genes Dev. 2008 Apr 1;22(7):872-877
- since 02/08
- Elite Network of Bavaria – Graduate school: Program: “Protein Dynamics in Health and Disease”
- 08/07 – 04/10
- Boehringer Ingelheim Fonds – PhD fellowship
- 04/07
- Roemer prize for young scientists: Award for Master’s thesis
- 11/06 – 12/06
- EMBO short-term fellowship: Support for Master’s thesis in Seattle, USA