Howard Hughes Medical Institute Investigator and Professor of Biochemistry, Biophysics and Structural Biology*
*And Affiliate, Division of Cell and Developmental Biology And Chair, Graduate Group in Biophysics
Our lab is dedicated to gaining mechanistic insight into crucial molecular processes in the life of the eukaryotic cell. Our two main research topics are the role of cytoskeletal dynamics in cell division, and the molecular mechanisms governing the flow of genetic information. The unifying principle in our work is the emphasis on studying macromolecular assemblies as whole units of molecular function via visualization of their functional states and regulatory interactions. We use electron microscopy (EM) and image analysis, biochemical and biophysical assays, towards a molecular understanding of our systems of interest.
Cytoskeleton and Cell Division
In our microtubule cytoskeleton studies we are interested in defining the conformational landscape of tubulin as defined by its nucleotide and assembly states, in order to obtain detail mechanistic understanding of the process of microtubule dynamic instability. Furthermore, to gain insight into how MT dynamics are regulated and utilized in the cellular context, we are studying the interactions of microtubule ends with cellular factors involved in chromosome capture, alignment and segregation. Our studies aim to provide fine details of the highly regulated interactions between microtubules and kinetochore complexes and other microtubule-interacting proteins essential for mitosis progression.
We have expanded our cytoskeletal studies to include the molecular understanding of septin assembly and function in cytokinesis. We have characterized the molecular architecture of septin assembly units, their polymerization into different assembly forms, and the relevance of their interaction with phospho-inositides. More recently we have moved into the realm of in vivo studies of the cytokinetic process in yeast. We are deepening our understanding of all of these aspects of septin structure and function and extending our studies to the characterization of septin interaction with cellular partners.
Gene Regulation and the Central Dogma
Our studies of nucleic acid transactions have been most powerful at defining the overall architecture of a large number of macromolecular assemblies involved in the critical initiation steps in DNA replication, RNA transcription and translation, for which no crystallographic information yet exist (e.g. ORC, TFIID, eIF3), as well as putting the crystal structures of essential elements in RNA processing and protein degradation in the context of fully functional complexes (e.g. RLC, proteosome).Our aim is to gain mechanistic insight that goes beyond overall architecture, by pushing resolution, describing conformational landscapes, and relating structural states to function via the analysis of interactions with ligands and regulatory factors.
Providing a mechanistic understanding of human transcription regulation is a major research objective in our lab. Regulated gene transcription in eukaryotes requires the assembly of a complex molecular machinery that includes general factors, activators, cofactor complexes and chromatin modifying and remodeling factors. We are interested in characterizing the structure of these different components and how they interact to regulate transcription. We have made important progress towards this goal with the description of the structural dynamics of human TFIID and its relevance for recognition of core promoter DNA. We have used an in vitro reconstituted system to study the stepwise assembly of human TBP, TFIIA, TFIIB, Pol II, TFIIF, TFIIE and TFIIH onto promoter DNA using cryo-electron microscopy. Our structural analyses provide pseudo-atomic models at various stages of transcription initiation that illuminate critical molecular interactions, including how TFIIF engages Pol II and promoter DNA to stabilize both the closed pre-initiation complex and the open-promoter complex, and to regulate start -site selection. Comparison of open versus closed pre-initiation complexes, combined with the localization of the TFIIH helicases XPD and XPB, support a DNA translocation model of XPB and explain its essential role in promoter opening.
Zhang, R., Alushin, G.M., Brown, A. and Nogales e. (2015) Mechanistic origin of microtubule dynamic instability and its regulation by EB proteins. Cell 162, 849-859.
Taylor, D.W., Zhu, Y., Staals, R.H.J., Kornfield, J.E., Shinkai, A., vander Oost, J., Nogales, E. and Doudna, J.A. (2015) Structures of the CRISPR-Cmr complex reveal mode of RNA target positioning. Science 348, 581-585.
Alushin, G.M., Lander, G.C., Kellogg, E.H., Zhang, R., Baker, D. and Nogales, E. (2014) High- resolution microtubule structrues reveal the structural transitions in ab-tubulin upon GTP hydrolysis. Cell 157, 1117,1129.
He, Y., Fang, J., Taatjes, D.J., and Nogales, E. (2013) Structural visualization of key steps in human transcription initiation. Nature 495, 481-486.
Cianfrocco, M.A., Kassevitis, G.A., Grob, P, Fang, J., Juven-Gershon, T., Kadonaga, J.T. and Nogales, E. (2013) Human TFIID binds core promoter DNA in a reorganized structural state. Cell 152, 120-131.
Ciferri, C., Lander, G.C., Maiolica, A., Herzog, F., Aebersold, R. and Nogales, E. (2012) Structure of the polycomb represive complex 2 and implications for gene silencing. eLIFE, e00005.
Lander, G.C., Estrin, E., Matyskiela, M.E., Bashore, C., Nogales, E. and Martin, A. (2012) Complete subunit architecture of the proteosome regulatory particle. Nature 482,186-191.
Garcia, G.III, Bertin, A., Li, Z., McMurray, M., Thorner, J. and Nogales, E. (2011) Subunit-dependent modulation of septin assembly: budding yeast septin Shs1 promotes ring and gauze formation. JCB 195, 993-1004.
Wiedenheft, B., Lander, G.C., Zhou, K., Jore, M.M., Brouns, S.J.J., van der Oost, J., Doudna, J.A., and Nogales, E. (2011) Structrues of the RNA-guided surveillance complex from a bacterial immune system. Nature 477, 486-489.
Alushin, G., Ramey, V.H., Pasqualato, S., Ball, D., Grigorieff, N., Musacchio, A. and Nogales, E. (2010) Visualization of sleeves of the NDC80 complex on microtubules. Nature 467, 805-810.
Bertin, A., McMurray, M.A., Grob, P., Park, S-S., Garcia, G. III, Patanwala, I., Ng, H-L., Alber, T.C., Thorner, J. and Nogales, E. (2008) Saccharomyces cerevisiae septins: Supramolecular organization of hetero-oligomers and the mechanism of filament assembly. PNAS 105, 8274-8279.
Wang, H-W., Ramey, V.H., Westermann, S., Leschziner, A.E., Welburn, J.P.I., Nakajima, Y., Drubin, D.G., Barnes, G. and Nogales, E. (2007) Architecture of the Dam1 kinetochore ring complex: implications for microtubule-driven assembly and force-coupling mechanisms. NSMB, 14, 721-726
Siridechadilok, B., Fraser, C.S., Hall, R.J., Doudna, J.A. and Nogales, E. (2005) Structural roles for human translation factor eIF3 in the initiation of protein synthesis. Science, 310, 1513-1515.
Wang, H-W. and Nogales, E. (2005) The nucleotide-dependent bending flexibility of tubulin regulates microtubule assembly. Nature, 435, 911-915
For a full publication listing please visit http://cryoem.berkeley.edu/pub
Photo credit: Mark Hanson at Mark Joseph Studios.
Last Updated 2015-09-07