W0007

Aminoacylation by Yeast Arginyl-tRNA Synthetase: Substrates Recognition and Specificity. Jean Cavarelli, Benedicte Delagoutte, Gilbert Eriani, Dino Moras, Laboratoire de Biologie Structurale, Institut de Genetique et de Biologie Moleculaire et Cellulaire, 1 rue Laurent Fries, BP 163, 67404 Illkirch, France

The biosynthesis of proteins from amino acids as coded by messenger RNA (mRNA) is done on the ribosomes and uses charged transfer RNAs (tRNAs) as key intermediates. The specific charging of a tRNA with its cognate amino acid is catalyzed by aminoacyl tRNA synthetase (aaRS), a family of 20 enzymes divided into two classes characterized by the topology of the active site. Correct charging is achieved by means of a two step catalytic reaction. The correct amino acid is first activated by ATP to form an aminoacyl-adenylate and then is transferred to the 3' terminal adenosine of the tRNA. Despite the structural and chemical similarities exhibed by the substrates, the accuracy of this enzymatic reaction is very high. Crystallographic studies of two class I synthetases (TrpRS and TyrRS) and four class II enzymes (AspRS, HisRS, LysRS,SerRS) have revealed the structural details of amino acid or adenylate specific recognition by these enzymes. Cytoplasmic ArgRS from yeast, a class I aaRS, is a monomer of 607 amino acid residues. Like GluRS and GlnRS, ArgRS needs to bind its cognate tRNA before it can form arginyl-adenylate from arginine and ATP. Yeast ArgRS has been cloned,overexpressed, purified and crystallized in presence of the arginine substrate. The structure has been solved by multiple isomorphous replacement and anomalous scattering combined with density modification methods and refined to 2.75 A (R=0.19, Rfree=0.25). The structure of ArgRS is built around a characteristic Rossmann fold to which unique domains are added or inserted. It shows the interactions made between arginine and its binding site which explain the specificity of ArgRS. Full details of the structure will be presented.