Probing ribosome structure by europium-induced RNA cleavage

Divalent metal ions are absolutely required for the structure and catalytic activities of ribosomes, They are partly coordinated to highly structured RNA, which therefore possesses high-affinity metal ion binding pockets. As metal ion induced RNA cleavages are useful for characterising metal ion bin ding sites and RNA structures, we analysed europium (Eu3+) induced specific cleavages in both 16S and 23S rRNA of E. coli. The cleavage sites were ide ntified by primer extension and compared to those previously identified for calcium, lead, magnesium, and manganese ions, Several Eu3+ cleavage sites, mostly those at which a general metal ion binding site had been already id entified, were identical to previously described divalent metal ions. Overa ll, the Eu3+ cleavages are most similar to the Ca2+ cleavage pattern, proba bly due to a similar ion radius. Interestingly, several cleavage sites whic h were specific for Eu3+ were located in regions implicated in the binding of tRNA and antibiotics. The binding of erythromycin and chloramphenicol, b ut not tetracycline and streptomycin, significantly reduced Eu3+ cleavage e fficiencies in the peptidyl transferase center, The identification of speci fic Eu3+ binding sites near the active sites on the ribosome will allow to use the fluorescent properties of europium for probing the environment of m etal ion binding pockets at the ribosome's active center.