Silvia Onesti
Head of Structural Biology
Higher Education
Degree in Chemistry, University of Pavia
PhD in Structural Biology, Imperial College London
Previous Appointments
Post-doctoral fellow in Prof. David Blow’s group at Imperial College
CNR Research Scientist, University of Pavia.
Lecturer, Dept. of Physics, Imperial College.
Senior Lecturer, Dept. of Biological Sciences, Imperial College.
Teaching
Professor of Structural Biology at SISSA, Trieste
(International School of Advanced Studies)
http://www.sissa.it/sbp/web_2008/C_Structural_Biology.html
Research
Structural biology is an interdisciplinary research area, requiring expertises from both the life sciences and the physical sciences. We apply molecular and structural biology tools to study the basic genetic processes within the cell, such as DNA replication and transcription. We use protein crystallography to determine the atomic structure of eukaryotic and archaeal proteins involved in these processes. Crystallographic studies are complemented by the concomitant use of electron microscopy to visualise the architecture of large complexes.
DNA replication and transcription are crucial event in the cell cycle, underpinning cellular processes with important consequences such as cell proliferation and genome stability. Failure to control these processes causes chromosome instability, which can lead to the development of cellular abnormalities, genetic disease and the onset of cancer.
Transcription
RNA polymerases (RNAP) are complex enzymes that contain a large catalytic core conserved from prokaryotes to human. In addition to this conserved unit, archaeal and eukaryotic polymerases also include a number of smaller polypeptides, most of which are absolutely required for transcription. We investigate the 3D structures of various subunits in order to learn more about RNA polymerase architecture.
We have determined the crystal structure of S. cerevisiae RPB5, one of the subunits shared by all three eukaryotic RNAPs (Todone et al., 2000) and the
the complex between the Methanococcus jannaschii, the archaeal homologues of Rpb7 and Rpb4 (Todone et al., 2001) and proposed a function for Rpb7 in binding the nascent RNA transcript. The structure suggested an intriguing and previously undetected homology between the RNA polymerase II Rpb4/Rpb7 complex and RNA polymerase I subunits A14 and A43 (Meka et al., 2003). We have also determined the structure of the human Rpb4/Rpb7 heterodimer and complemented the structural analysis with biochemical studies directed at dissecting the RNA binding properties of the complex (Meka et al., 2005).
DNA replication
Although our understanding of eukaryotic DNA replication has improved considerably in recent years, the detailed mechanism of initiation of the reaction is still not known.
MCM proteins are large macromolecular assemblies acting as the replicative helicases and playing a key role in the initiation of DNA replication. We have obtained an initial three-dimensional reconstruction from negatively stained MCM particles from M. thermoautotrophicum (Pape et al., 2003). We have assessed the changes in stoichiometry that the complex undergoes when treated with various substrates (Costa et al., 2006a). 3D reconstructions were carried out for a dsDNA treated and an ADP.AlFx treated sample, respectively assembling as double hexamer and double heptamer (Costa et al., 2006b). The electron density maps display an unexpected asymmetry, between the two rings, suggesting that large conformational changes can occur within the complex.
We have visualized a novel interaction between MCM and dsDNA, with the DNA wrapping around the N-terminal face of a single hexameric ring. This interaction requires a conformational change within the outer belt of the MCM N-terminal domain, exposing a previously unrecognized helix-turn-helix DNA-binding motif. We suggest that this represents an initial site of interaction, prior to the loading and activation of the complex to function as a helicase at the fork (Costa et al., 2008)
Contact information
Sincrotrone Trieste S.C.p.A.
SS 14 - km 163,5 - AREA Science Park
34149 Basovizza, Trieste ITALY
Email: silvia.onesti@elettra.trieste.it
Tel. +39 040 3758451
Mob +39 366 6878001
Selected publications
Structural biology of MCM helicases.
Costa A. and Onesti S. (2009). Crit. Rev. Biochem. Mol. Biol. 44, 326-342.
Cryo-electron microscopy reveals a novel DNA binding site on the MCM helicase.
Costa A., Van Dujinen G., Medagli B., Chong J., Sakakibara N., Kelman Z., Nair S.K., Patwardhan A. and Onesti S. (2008). EMBO J. 27, 2250-2258.
Structural basis of the Methanobacter thermautotrophicus MCM helicase activity.
Costa A., Pape T., van Heel M., Brick P., Patwardhan A. and Onesti S. (2006)
Nucleic Acid Res. 34, 5829-5838.
The Elongator subunit Elp3 contains a Fe4S4 cluster and binds S-adenosylmethionine.
Paraskevopoulou C., Fairhurst S.A., Lowe D.J., Brick P. and Onesti S. (2006)
Mol. Microbiol. 59, 795-806.
Crystal structure and RNA binding of the Rpb4/Rpb7 subunits of human RNA polymerase II.
Meka H., Werner F., Cordell, S., Onesti S. and Brick P. (2005)
Nucleic Acid Res. 33, 6435-6444.
Hexameric ring structure of the full-length archaeal MCM complex.
Pape T., Meka H., Chen S., Vicentini G., van Heel M. and Onesti S. (2003)
EMBO Rep. 4, 1079-1083.
Structural and functional homology between the RNAPI subunits A14/A43 and the archaeal RNAP subunits E/F.
Meka H., Daoust G., Bourke-Arnvig K., Werner F., Brick P. and Onesti S. (2003)
Nucleic Acid Res. 31, 4391-4400.
Structure of an archaeal homologue of the eukaryotic RNA polymerase II RPB4/RPB7 complex.
Todone F., Brick P., Werner, F., Weinzierl R.O.J and Onesti S. (2001)
Mol. Cell, 8, 1137-1143.
Crystal structure of RPB5, a universal eukaryotic RNA polymerase subunit and transcription factor interaction target.
Todone F., Weinzierl R.O.J, Brick P. and Onesti S. (2000)
Proc. Natl. Acad. Sci. USA, 97, 6306-6310.
