RNA SEQUENCE DETERMINANTS FOR AMINOGLYCOSIDE BINDING TO AN A-SITE RIBOSOMAL-RNA MODEL OLIGONUCLEOTIDE

The codon-anticodon interaction on the ribosome occurs in the A site o f the 30 S subunit. Aminoglycoside antibiotics, which bind to ribosoma l RNA in the A site, cause misreading of the genetic code and inhibit translocation. Biochemical studies and nuclear magnetic resonance spec troscopy were used to characterize the interaction between the aminogl ycoside antibiotic paromomycin and a small model oligonucleotide that mimics the A site of Escherichia coli 16 S ribosomal RNA. Upon chemica l modification, the RNA oligonucleotide exhibits an accessibility patt ern similar to that of 16 S rRNA in the 30 S subunit. In addition, the oligonucleotide binds specifically aminoglycoside antibiotics. The an tibiotic binding site forms an asymmetric internal loop, caused by non -canonical base-pairs. Nucleotides that are important for binding of p aromomycin were identified by performing quantitative footprinting on oligonucleotide sequence variants and include the C1407 . G1494 base-p air, and A . U base-pair at positions 1410/1490, and nucleotides A1408 , A1493 and U1495. The asymmetry of the internal loop, which requires the presence of a nucleotide in position 1492, is also crucial for ant ibiotic binding. Introduction into the oligonucleotide of base changes that are known to confer aminoglycoside resistance in 16 S rRNA resul t in weaker binding of paromomycin to the oligonucleotide. Oligonucleo tides homologous to eukaryotic rRNA sequences show reduced binding of paromomycin, suggesting a physical origin for the species-specific act ion of aminoglycosides. (C) 1996 Academic Press Limited