Design of RNA domains, substrates or inhibitors of tRNA-recognising proteins

This project concerns investigations on RNA domains designed to interact with- or to inhibit proteins specific of transfer ribonucleic acids. The RNAs we will investigate derive from cannoncial tRNA (taken as a model) or viral tRNA-like structures, either by rational structure-based design or by in vitro selection procedures. The final aim is to find small RNA molecules that specifically block "t-RNA"-target proteins (tRNA maturation endonucleases, aminoacyl-tRNA synthetases, elongation factors, viral replicases). Static and dynamic RNA structures will be investigated together with RNA interaction modes with proteins or other ligands such as metal ions. Various structural biology approaches (X-ray crystallography, NMR, chemical probing) will be employed in synergy with molecular biology (RNA engineering, combinatorial methods, enzymology), chemistry (synthesis of RNA), and theory (computational biology, structure predictions). We are interested in understanding detailed molecular aspects of the translation machinery. Part of the project concerns transcription / replication, since tRNA or tRNA-like domains participate in such processes. Emphasis will be given to studies on functional small RNA domains and to comparsion with features characterising the corresponding full-length natural RNAs. In this way basic properties of the RNAs will be revealed and full use of NMR or chemical synthesis can be made. In our program we intend to study a variety of steps in translation (and some in replication) and to work on RNAs originating from various biological backgrounds (viruses, prokaryotes - E. coli & thermophiles-, eukaryotes yeast, archaea) in order to understand species specificities and to learn about evolutionary relationships between these RNAs, in particular for aminoacylation and elongation. The problem of evolution will be approached by theoretical methods for the search of RNAs with tRNA characteristic and by in vitro selection procedures. In this way we expect to find RNA structures similar to those retained by natural evolution, but we hope also to find alternate solutions providing aminoacylation identities or recognition potential by translation factors. Once the RNA / protein recognition modes understood, it will be relatively easy to introduce the appropriate mutations in these RNAs that will prevent their functions. The feasibility of the project relies on the complementary expertises in RNA research of the 7 participainting Teams (including 1 Industrial Partner) both in macromolecular engineering methods and in knowledge of biologicalsystems. The project will generate a synergy not possible in the iondividual Laboratories. Thus, features and rules important for understanding the large RNA molecules, and will provide the basic knowledge for the biotechnological applications (e.g. engineering of the translational machinery, species specific control of particular steps in this machinery, design of RNA aptamers with antibiotic functions, …).