Research Overview

The protein content of the cell strongly determines what a cell is and how it will react to its external environment. The ability of the cell to ensure and orchestrate the coordinated appearance of a required functional protein is, therefore, essential for its own existence. Protein synthesis in all living cells, takes place on the ribosome, a macromolecular complex that totals, in E. coli, 2.3 MDa. Because of its fundamental role for the cell, the translational apparatus is strictly regulated. Indeed a large number of translation factors, chaperones, and assembly factors have been identified that modulate gene expression by binding to and interacting with the ribosome. Furthermore, the fundamental nature of the translation makes the ribosome one of the major targets for antibiotic inhibition.

Using X-ray crystallography (within our group) or CryoEM and NMR (in collaborative studies), the main aim of our research is to understand from a structural point of view how the various components of the translational apparatus interact with each other to guide and regulate ribosome activity or to ensure that the de-novo synthesized polypeptide nascent chain acquires its functional structure. 

Our research projects encompass four main research areas:

1) Factors involved in the very basic steps of protein synthesis, such as the translocation phase. In a collaborative study with Prof. Christian Spahn (Berlin, Germany), we have been interested studying studying the binding mode and action of EF-G. This  translational factor directs the movement of the ribosome along the mRNA, to enable at each step of the elongation cycle the incorporation of a new amino acid in the growing polypeptide chain (Connell et al., 2007 Molecular Cell). Complementary to these studies, we structurally analysed LepA, a translational factor able to revert the translocation step (Connell et al, 2008, Molecular Cell). 

2) Ribosome Assembly is the delicate step of orchestrating the biogenesis of the ribosome where more than 55 ribosomal proteins and three nucleic acid strands composed by more than 4500 nucleotides, have to assemble together in a unique conformation to constitute the active ribosome. An example of our studies in this field are the collaborative projects with Prof. Rajandra Agrawal (Albany, USA) that have revealed the binding mode and likely role of Era - a small GTPase - and the cold shock protein RbfA, in the assembly of the small ribosomal subunit (Datta et al, Molecular Cell 2007; Sharma et al., Molecular Cell 2005).  

3) The crystal structure of various antibiotics in complex with the ribosome is also at the centre of our studies. These projects aim to understand how these small ligands, usually one thousand times smaller than the ribosome, are able to knock out completely the activity of such a large macromolecular complex. Among the inhibitors studied, thiostrepton and micrococcin which, interestingly, although belonging to the same class of antibiotics and sharing the same ribosomal binding pocket, are able to block the ribosome in two distinct conformations resembling its ‘off’ and ‘on’ states (Harms et al., Molecular Cell 2008).

4) The cross-talk between the nascent chain and the ribosome, and co-translational folding. The most challenging projects ongoing in the group concerns the folding acquisition of the polypeptide nascent chain. Synthesised in the core of the ribosome, the nascent chain has to pass a long conduit, of c.a. 100 Å to reach the outside of the particle. This passage is emerging as a highly dynamic and 'personalized' event, in which, a continuous cross-talk exists between the ribosome and the translated nascent chain. As a result, the ribosome seems to be able to recognize the protein it is synthesizing and accordingly regulate the speed of translation and organize its correct compartmentalization. In a joint effort with the groups of Dr. Takemoto (Yokohama, Japan) Dr. Christodoulu (London, UK), Prof. Dobson (Cambridge, UK) and Dr. Sean Connell (Frankfurt, Germany) we are trying to obtain, at a structural level, a detailed description of the polypeptide chain, while being translated on the ribosome and thereby elucidate its co-translational folding, its interaction with the ribosome and the mechanism by which these interactions regulate protein synthesis. The structural approaches used - X-ray crystallography, NMR (Hsu et al., PNAS 2007) and cryoEM – should allow a unique understanding of these fundamental aspects of the translational machinery for which, to date, no atomic details are available for any of the events described. The projected results will increase our basic understanding of the translational process and create the basis for the development of more efficient translation systems and new antibiotics. Furthermore, in light of the emerging range of protein misfolding disease, the understanding of the folding properties in the context of translation could offer new directions for the treatments of such diseases.