Specific metal ion binding sites in rRNA and the influence of metal ions on peptide bond formation

Ribosomes are central to the biosynthesis of proteins and are therefore an important target for antibiotics. This RNP particle translates the genetic information transported by the mRNA into the amino acid sequence of the proteins. The enzymatic activity (peptidy transferase) which catalyzes peptide bond formation is localized on the large subunit of the ribosome. As evidences have accumulated that ribosomal RNA and not proteins are responsible for the catalytic activity, we have recently developed a model proposing that peptide bond formation is catalyzed by metal ions. It is known that the integrity of the ribosomes as well as all functional aspects are crucially dependent on magnesium ions, but only little is known about specific metal binding sites in this important enzymatic complex. The aim of this project is to characterize metal binding sites in ribosome in more detail and to investigate whether metal ions are involved in the catalysis of peptide bond formation. Metal ion induced cleavages in rRNA and tRNA will be used to probe both RNA structures during the ribosomal cycle. Furthermore, this method will be used to study metal ion binding pockets in rRNA and to reconstruct metal binding sites from in vivo and in vitro synthesized RNA. This metal ion binding sites will be investigated in detail. To falsify the hypothesis of a metal catalyzed peptide bond formation thio-substitued P-site substrates will be synthesized and used for ,,metal ion rescue" experiments. The results of these experiments will advance our understanding on metal ion binding sites in ribosome and on the everlasting question how peptide bond formation is catalyzed.

Ribosomes are complex RNP particles where the mRNA dependent protein synthesis takes place. Several activities have to be coordinated on this large complex, namely the correct binding of mRNA, the positioning of the tRNAs and peptide bond formation. During translocation, the tRNA/mRNA complex has to be shifted for a triplet codon to allow the next amino acid to be incorporated into the growing peptide chain. These recurrent dynamic steps are fuelled by GTP but the moving parts of this translational machine are not defined. The structural integrity of the ribosomes as well as all functional aspects are crucially dependent on magnesium ions. This project has investigated the significance of metal ions for catalysis of peptide bond formation and has used the very sensitive method of metal ion cleavage for determining structural changes in the rRNA during translocation. Indeed, during translocation structural changes have been observed in the 23S RNA in close vicinity of the active site of peptide bond formation. We could also monitor these recurrent changes in the RNA after each addition of an amino acid to the growing peptide chain. Furthermore, we could determine pH dependent structural changes at the peptidyl transferase center which conforms to the observed pH enhancement of peptide bond formation. Experiments with small modified P-site stubstrates have identified the adjacent hydroxyl group as an important factor for the reactivity of the amino acyl ester, possibly influenced by a metal ion. This investigations contribute to the ongoing debate on the catalysis of peptide bond formation and might also give insight into the function of some antibiotics.