We are interested in understanding how RNA helicases work, how their specificity and regulation are conferred, and why they play such important roles in the cell. We concentrate on the DEAD-box proteins, and we typically use yeast and bacteria as model systems. We are interested in characterizing how the conserved “helicase” core works. We use a triad of approaches. First, we use sequence alignments and molecular modeling to identify regions of interest. Then we use bacterial and yeast genetics to assay the effects of deletions and site-specific mutations in vivo. Finally, we use purified recombinant proteins, expressed in bacteria, to characterize their enzymatic properties. These include their RNA-dependent ATPase activity, their RNA binding affinities and their capacity to displace RNA-RNA and RNA-DNA duplexes in vitro. We use various DEAD-box proteins in order to elucidate common features, but we concentrate on Ded1 from yeast because it is easy to work with and because it has one of the highest activities for a DEAD-box protein. It can easily dissociate a 50-fold excess of an 18 base pair RNA-DNA duplex in under 5 minutes, with a rate of ATP hydrolysis of 350 molecules ATP per Ded1 per minute.

However, in vitro Ded1 lacks substrate specificity and enzymatic regulation that must exist in vivo.
  Thus, we also are interested in identifying the protein partners and RNA substrates of Ded1. We use pull-down experiments of cellular extracts to identify factors interacting with Ded1 in vivo that we subsequently characterize in vitro with purified recombinant proteins. We use fusion proteins with fluorescent tags to colocalize Ded1 with its partners in living cells under different conditions in collaboration with Dr. Naïma Belgareh-Touzé, IBPC.

Finally, we are collaborating with Dr. Ikram Guizani, Pasteur Institute Tunis, to screen for biologically active agents specific for the DEAD-box protein LeIF from the parasitic trypanosomatid protozoa Leishmania. Dr. Michael Nilges, Pasteur Institute Paris, heads the unit modeling the three-dimensional structure of LeIF and identifying potential targets for therapeutic agents, while Dr. Hélène Munier-Lehmann, Pasteur Institute Paris, is involved in large-scale screening of potential agents. (Kyle Tanner)

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Our work also focuses on the methylation of the translational machinery. Our model organism is S. cerevisiae, which is an extraordinarily powerful genetic system with a well-defined translation system. (Valérie Heurgué-Hamard)

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