Fluorescence-based approach for detecting and characterizing antibiotic-induced conformational changes in ribosomal RNA: comparing aminoglycoside binding to prokaryotic and eukaryotic ribosomal RNA sequences.
Aminoglycoside antibiotics bind specifically to a conserved sequence of the 16S ribosomal RNA (rRNA) A site and interfere with protein synthesis. One model for the mechanism underlying the deleterious effects of aminoglycosides on protein synthesis invokes a drug-induced conformational change in the rRNA that involves the destacking of two adenine residues (A1492 and A1493 in Escherichia coli) at the A site. We describe here a fluorescence-based approach for detecting and characterizing this drug-induced conformational change in the target rRNA. In this approach, we insert the fluorescent base analogue 2-aminopurine in place of A1492 in an E. coli 16S rRNA A-site model oligonucleotide (EcWT) as well as in a mutant form of this oligomer (A1408G) in which A1408 has been replaced with a guanine. The presence of guanine at 1408 instead of adenine represents one of the major sequence differences between prokaryotic and eukaryotic A sites, with the latter A sites being resistant to the deleterious effects of aminoglycosides. Binding of the aminoglycoside paromomycin to the 2AP-substituted forms of EcWT and A1408G induced changes in fluorescence quantum yield consistent with drug-induced base destacking in EcWT but not A1408G. Isothermal titration calorimetry studies reveal that paromomycin binds to the EcWT duplex with a 31-fold higher affinity than the A1408G duplex, with this differential affinity being enthalpic in origin. In the aggregate, these observations are consistent with both rRNA binding affinity and drug-induced base destacking being important determinants in the prokaryotic specificity of aminoglycosides. Combining fluorescence quantum yield and lifetime data allows for quantification of the extent of drug-induced base destacking, thereby providing a convenient tool for evaluating the relative impacts of both novel and existing A-site targeting ligands on rRNA conformation and potentially for predicting relative antibiotic activities and specificities.
Kaul
Malvika
M
Department of Pharmacology, University of Medicine and Dentistry of New Jersey-Robert Wood Johnson Medical School, 675 Hoes Lane, Piscataway, New Jersey 08854-5635, USA.
Barbieri
Christopher M
CM
Pilch
Daniel S
DS
D015075:2-Aminopurine / Q000737:chemistry; D000900:Anti-Bacterial Agents / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology*; D004926:Escherichia coli / Q000187:drug effects / Q000235:genetics; D007700:Kinetics; D008958:Models, Molecular; D009690:Nucleic Acid Conformation / Q000187:drug effects; D010303:Paromomycin / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology*; D012336:RNA, Ribosomal, 16S / Q000737:chemistry / Q000187:drug effects* / Q000378:metabolism; D013050:Spectrometry, Fluorescence / Q000379:methods*; D013056:Spectrophotometry, Ultraviolet; D013379:Substrate Specificity; D013816:Thermodynamics
D000900:Anti-Bacterial Agents; D012336:RNA, Ribosomal, 16S; D015075:2-Aminopurine; D010303:Paromomycin