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Structural and Computational Biology Unit Molecular mechanisms of transcriptional regulation and epigenetics Previous and current research Our group is interested in molecular mechanisms of transcriptional regulation in eukaryotes, where DNA is packaged into chromatin. In the context of chromatin, we are interested how different sequence-specific transcription factors assemble on DNA and how sequence-specific transcription factors interact with co-activators and general transcription factors to recruit RNA polymerases to the transcription start site. We are also studying the overall structure, architecture and inner workings of large molecular machines like RNA polymerases or remodelling factors involved in the transcription process. Finally, we like to gain insight how DNA sequence information and epigenetic modifications act together to regulate gene transcription. To achieve these goals, we use structural information obtained by X-ray crystallography and electron microscopy combined with other biophysical and biochemical techniques. Systems currently under investigation include transcription factor/DNA complexes, yeast RNA polymerase III and multi-protein complexes involved in chromatin targeting, remodelling and histone modifications. RNA polymerase III consists of 17 subunits and is responsible for the transcription of small RNAs like tRNA and 5S RNA. Recruitment of the enzyme requires binding of the general transcription factor TFIIIC, composed of six subunits, to internal promoter sites followed by the binding of TFI- IIB composed of three subunits. Our research aims to understand the overall architecture of RNA polymerase III, TFIIIC and TFIIIB and their interactions during the RNA polymerase III recruitment process, transcriptional elongation and termination. Christoph W. Müller PhD 1991, University of Freiburg. Postdoctoral work at Harvard University, Cambridge, Massachusetts. At EMBL Grenoble since 1995. Joint Unit Coordinator at EMBL Heidelberg since 2007. Joint appointment with the Gene Expression Unit. The accessibility of chromatin in eukaryotes is regulated by ATP-dependent chromatin remodelling factors and histone-modifying enzymes. Both classes of enzymes use similar domains like bromodomains, chromodomains, MBT domains, PHD fingers and SANT domains for the controlled access to defined genomic regions. Many of these enzymes also do not work in isolation, but rather in the context of larger multi-subunit assemblies. We try to understand the molecular architecture of chromatin modifying and remodelling complexes, by which mechanisms they are recruited, how they interact with chromatin templates and how their activities are regulated. Future projects and goals • Molecular insights into the recruitment of transcriptional regulator through the combination of DNA sequence-specific recognition and epigenetic modifications. Figure 1 (above): Model of transcribing RNA polymerase III as determined by single particle electron microscopy. The incoming DNA is depicted in blue and white, while the transcribed RNA is depicted in red. • Structural and functional analysis of macromolecular machines involved in transcription, chromatin remodelling and chromatin modification. • Contributing to a better mechanistic understanding of eukaryotic transcription and epigenetics using biochemical and structural biology approaches. Figure 2 (right): Crystal structure of the τ60/∆τ91 subcomplex of yeast general transcription factor IIIC. Selected references Hamès, C., Ptchelkine, D., Grimm, C., Thevenon, E., Moyroud, E., Gérard, F., Martiel, J.L., Benlloch, R., Parcy, F., Müller, C.W. (2008). Structural basis for LEAFY floral switch function and similarity with helix-turn-helix proteins. EMBO J., 27, 2628-37 Fernandez-Tornero, C., Böttcher, B., Riva, M., Carles, C., Steuerwald, U., Ruigrok, R.W., Sentenac, A., Müller, C.W., Schoehn, G. (2007). Insights into transcription initiation and termination from the electron microscopy structure of yeast RNA polymerase III. Mol. Cell, 25, 813-23 Grimm, C., de Ayala Alonso, A.G., Rybin, V., Steuerwald, U., Ly- Hartig, N., Fischle, W., Müller, J., Müller, C.W. (2007). Structural and functional analyses of methyl-lysine binding by the malignant brain tumour repeat protein Sex comb on midleg. EMBO Rep., 8, 1031-7 Mylona, A., Fernandez-Tornero, C., Legrand, P., Haupt, M., Sentenac, A., Acker, J., Müller, C.W. (2006). Structure of the τ60/Δτ91 subcomplex of yeast transcription factor IIIC: insights into pre-initiation complex assembly. Mol. Cell, 2, 221-232. 3
EMBL Research at a Glance 2009 John Briggs PhD 200, Oxford University. Postdoctoral research at the University of Munich. Group leader at EMBL since 2006. How proteins manipulate membranes – cryo-electron microscopy and tomography Previous and current research A cell’s control over the shape and dynamics of its membrane systems is fundamental to its function. We are interested in how proteins can define and manipulate the shapes of membranes during budding and fusion events. To explore this question we are studying a range of different cellular and viral specimens using cryo-electron microscopy and tomography. A particular emphasis of our research is the structure and life-cycle of asymmetric membrane viruses such as HIV. The assembly of the virus particles and their subsequent fusion with target cells offer insights into general features of vesicle budding and membrane fusion. Cryo-electron microscopy techniques are particularly appropriate for studying vesicles and viruses because they allow membrane topology to be observed in the native state, while maintaining information about the structure and arrangement of associated proteins. Computational image processing and three-dimensional reconstructions are used to extract and interpret this information. We take a step-by-step approach to understanding the native structure. Three dimensional reconstructions can be obtained using cellular cryoelectron tomography of the biological system in its native state. These reconstructions can be better interpreted by comparison with data collected from in vitro reconstituted systems. A detailed view is obtained by fitting these reconstructions with higher resolution structures obtained using cryo-electron microscopy and single particle reconstruction of purified complexes. Future projects and goals Our goal is to understand the interplay between protein assemblies and membrane shape. How do proteins induce the distortion of cellular membranes into vesicles of different dimensions? What are the similarities and differences between the variety of cellular budding events? How do viruses hijack cellular systems for their own use? What is the role and arrangement of the cytoskeleton during membrane distortions? What membrane topologies are involved in fusion of vesicles with target membranes? How does the curvature of a membrane influence its interaction with particular proteins? We will develop and apply microscopy and image processing approaches to such questions. Figure 2 (below): 3D reconstruction of the SIV glycoprotein spike, generated by averaging sub-tomograms extracted from whole virus tomograms. (Zanetti et al., 2006) Figure 1: 3D reconstruction of HIV-1 virions using cryoelectron microscopy. Selected references Carlson, L.A., Briggs, J.A., Glass, B., Riches, J.D., Simon, M.N., Johnson, M.C., Muller, B., Grunewald, K. & Krausslich, H.G. (2008). Three-dimensional analysis of budding sites and released virus suggests a revised model for HIV-1 morphogenesis. Cell Host Microbe, , 592-599 Briggs, J.A., Grunewald, K., Glass, B., Forster, F., Krausslich, H.G. & Fuller, S.D. (2006). The mechanism of HIV-1 core assembly: insights from 3D reconstructions of authentic virions. Structure, 1, 15-20 Briggs, J.A., Johnson, M.C., Simon, M.N., Fuller, S.D. & Vogt, V.M. (2006). Cryo-electron microscopy reveals conserved and divergent features of gag packing in immature particles of Rous sarcoma virus and human immunodeficiency virus. J. Mol. Biol., 355, 157-168 Zanetti, G., Briggs, J.A., Grunewald, K., Sattentau, Q.J. & Fuller, S.D. (2006). Cryo-electron tomographic structure of an immunodeficiency virus envelope complex in situ. PLoS Pathog., 2, e83
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