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 binding sites and RNA structures, we analysed europium (Eu3+) induced specific cleavages in both 16S and 23S rRNA of E. coli. The cleavage sites were identified 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 identified, were identical to previously described divalent metal ions. Overall, the Eu3+ cleavages are most similar to the Ca2+ cleavage pattern, probably due to a similar ion radius. Interestingly, several cleavage sites which were specific for Eu3+ were located in regions implicated in the binding of tRNA and antibiotics. The binding of erythromycin and chloramphenicol, but not tetracycline and streptomycin, significantly reduced Eu3+ cleavage efficiencies in the peptidyl transferase center. The identification of specific Eu3+ binding sites near the active sites on the ribosome will allow to use the fluorescent properties of europium for probing the environment of metal ion binding pockets at the ribosome's active center.
Dorner
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Institut für Biochemie, Universität Wien, Vienna Biocenter, Austria.
Barta
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D000900:Anti-Bacterial Agents / Q000378:metabolism; D001483:Base Sequence; D002412:Cations; D002701:Chloramphenicol / Q000378:metabolism; D004917:Erythromycin / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D005063:Europium / Q000378:metabolism*; D008969:Molecular Sequence Data; D012329:RNA, Bacterial / Q000378:metabolism; D012335:RNA, Ribosomal / Q000737:chemistry / Q000378:metabolism*; D012336:RNA, Ribosomal, 16S / Q000737:chemistry / Q000378:metabolism*; D012270:Ribosomes; D013307:Streptomycin / Q000378:metabolism
D000900:Anti-Bacterial Agents; D002412:Cations; D012329:RNA, Bacterial; D012335:RNA, Ribosomal; D012336:RNA, Ribosomal, 16S; D005063:Europium; D004917:Erythromycin; D002701:Chloramphenicol; D013307:Streptomycin