RNA sequence determinants for aminoglycoside binding to an A-site rRNA model oligonucleotide.
The codon-anticodon interaction on the ribosome occurs in the A site of the 30 S subunit. Aminoglycoside antibiotics, which bind to ribosomal RNA in the A site, cause misreading of the genetic code and inhibit translocation. Biochemical studies and nuclear magnetic resonance spectroscopy were used to characterize the interaction between the aminoglycoside antibiotic paromomycin and a small model oligonucleotide that mimics the A site of Escherichia coli 16 S ribosomal RNA. Upon chemical modification, the RNA oligonucleotide exhibits an accessibility pattern similar to that of 16 S rRNA in the 30 S subunit. In addition, the oligonucleotide binds specifically aminoglycoside antibiotics. The antibiotic binding site forms an asymmetric internal loop, caused by non-canonical base-pairs. Nucleotides that are important for binding of paromomycin were identified by performing quantitative footprinting on oligonucleotide sequence variants and include the C1407.G1494 base-pair, 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 antibiotic binding. Introduction into the oligonucleotide of base changes that are known to confer aminoglycoside resistance in 16 S rRNA result in weaker binding of paromomycin to the oligonucleotide. Oligonucleotides homologous to eukaryotic rRNA sequences show reduced binding of paromomycin, suggesting a physical origin for the species-specific action of aminoglycosides.
Department of Chemistry and Biochemistry, University of California, Santa Cruz 95064, USA.
M I
MI
Recht
D
D
Fourmy
S C
SC
Blanchard
K D
KD
Dahlquist
J D
JD
Puglisi
D000617:Aminoglycosides / Q000378:metabolism*; D001345:Autoradiography; D018983:DNA Footprinting; D004352:Drug Resistance, Microbial / Q000235:genetics; D009682:Magnetic Resonance Spectroscopy; D009690:Nucleic Acid Conformation; D010303:Paromomycin / Q000378:metabolism; D012329:RNA, Bacterial / Q000737:chemistry*; D012335:RNA, Ribosomal / Q000737:chemistry / Q000378:metabolism*; D012336:RNA, Ribosomal, 16S / Q000737:chemistry; D013045:Species Specificity; D013329:Structure-Activity Relationship
D000617:Aminoglycosides; D012329:RNA, Bacterial; D012335:RNA, Ribosomal; D012336:RNA, Ribosomal, 16S; D010303:Paromomycin