Through an affinity chromatography based modification-interference assay, we have identified chemical groups within Escherichia coli 16S ribosomal RNA sequence that are required for binding the aminoglycoside antibiotic paromomycin. Paromomycin was covalently linked to solid support via a nine atom spacer from the 6′′′-amine of ring IV, and chemical modifications to an A-site oligonucleotide that disrupted binding were identified. Positions in the RNA oligonucleotide that correspond to

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... , , as well as the pro-R phosphate oxygens of A1492 and A1493 in 16S rRNA are chemical groups that are essential for a high-affinity RNA-paromomycin interaction. These data are consistent with genetic, biochemical, and structural studies related to neomycin-class antibiotics and provide additional information for establishing an exact model for their interaction with the ribosome. S0006-2960(97)03125-5 CCC: $15.00