Aminoglycoside antibiotics, including paromomycin, neomycin and gentamicin,
target a region of highly conserved nucleotides in the decoding region ami
noacyl-tRNA site (A site) of 16 S rRNA on the 30 S subunit. Change of a sin
gle nucleotide, A1408 to G, reduces the affinity of many aminoglycosides fo
r the ribosome; G1408 distinguishes between prokaryotic and eukaryotic ribo
somes. The structures of a prokaryotic decoding region A-site oligonucleoti
de free in solution and bound to the aminoglycosides paromomycin and gentam
icin Cia were determined previously. Here, the structure of a eukaryotic de
coding region A-site oligonucleotide bound to paromomycin has been determin
ed using NMR spectroscopy and compared to the prokaryotic A-site-paromomyci
n structure. A conformational change in three adenosine residues of an inte
rnal loop, critical for high-affinity antibiotic binding, was observed in t
he prokaryotic RNA-paromomycin complex in comparison to its free form. This
conformational change is not observed in the eukaryotic RNA-paromomycin co
mplex, disrupting the binding pocket for ring I of the antibiotic. The lack
of the conformational change supports footprinting and titration calorimet
ry data that demonstrate approximately 25-50-fold weaker binding of paromom
ycin to the eukaryotic decoding-site oligonucleotide. Neomycin, which is mu
ch less active against Escherichia coli ribosomes with an A1408G mutation,
binds non-specifically to the oligonucleotide. These results suggest that e
ukaryotic ribosomal RNA has a shallow binding pocket for aminoglycosides, w
hich accommodates only certain antibiotics. (C) 2001 Academic Press.