RNA Sequence Determinants for Aminoglycoside Binding to an A-site rRNA Model Oligonucleotide

Recht, Michael I.; Fourmy, Dominique; Blanchard, Scott C.; Dahlquist, Kam D; Puglisi, Joseph D.

doi:10.1006/jmbi.1996.0526

Cited by 246 publications

(221 citation statements)

References 43 publications

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“…More specifically, they display high affinities with certain domains of prokaryotic rRNA (31,32,33,44). The flexibility of the aminoglycosides allows their adaptation to the shape of the binding pocket in the internal loops of RNA helix cores to make specific contacts (12,45,47).…”

Section: Discussionmentioning

confidence: 99%

Plasmid-Mediated High-Level Resistance to Aminoglycosides in Enterobacteriaceae Due to 16S rRNA Methylation

Galimand

Courvalin

Lambert

2003

Antimicrob Agents Chemother

312

221

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A self-transferable plasmid of ca. 80 kb, pIP1204, conferred multiple-antibiotic resistance to Klebsiella pneumoniae BM4536, which was isolated from a urinary tract infection. Resistance to ␤-lactams was due to the bla TEM1 and bla CTX-M genes, resistance to trimethroprim was due to the dhfrXII gene, resistance to sulfonamides was due to the sul1 gene, resistance to streptomycin-spectinomycin was due to the ant3؆9 gene, and resistance to nearly all remaining aminoglycosides was due to the aac3-II gene and a new gene designated armA (aminoglycoside resistance methylase). The cloning of armA into a plasmid in Escherichia coli conferred to the new host high-level resistance to 4,6-disubstituted deoxystreptamines and fortimicin. The deduced sequence of ArmA displayed from 37 to 47% similarity to those of 16S rRNA m 7 G methyltransferases from various actinomycetes, which confer resistance to aminoglycoside-producing strains. However, the low guanine-pluscytosine content of armA (30%) does not favor an actinomycete origin for the gene. It therefore appears that posttranscriptional modification of 16S rRNA can confer high-level broad-range resistance to aminoglycosides in gram-negative human pathogens.

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Section: Discussionmentioning

confidence: 99%

Plasmid-Mediated High-Level Resistance to Aminoglycosides in Enterobacteriaceae Due to 16S rRNA Methylation

Galimand

Courvalin

Lambert

2003

Antimicrob Agents Chemother

312

221

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“…Ribosomal A Site-Sequence specificity of aminoglycoside antibiotics (especially paromomycin) binding to the prokaryotic ribosomal A site has been extensively studied by mutagenesis and footprinting analysis (23)(24)(25) and by surface plasmon resonance (26). First insights of the A site complexed with antibiotics were obtained by NMR (27,28).…”

Section: Sequence and Structure Similarities Between The Dis And Thementioning

confidence: 99%

HIV-1 RNA Dimerization Initiation Site Is Structurally Similar to the Ribosomal A Site and Binds Aminoglycoside Antibiotics

Ennifar

Paillart

Marquet

et al. 2003

Journal of Biological Chemistry

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Human immunodeficiency virus (HIV) genomic RNA is packaged into virions as a dimer. The first step of dimerization is the formation of a kissing-loop complex at the so-called dimerization initiation site (DIS). We found an unexpected and fortuitous resemblance between the HIV-1 DIS kissing-loop complex and the eubacterial 16 S ribosomal aminoacyl-tRNA site (A site), which is the target of aminoglycoside antibiotics. Similarities exist not only at the primary and secondary structure level but also at the tertiary structure level, as revealed by comparison of the respective DIS and A site crystal structures. Gel shift, inhibition of lead-induced cleavage, and footprinting experiments showed that paromomycin and neomycin specifically bind to the kissing-loop complex formed by the DIS, with an affinity and a geometry similar to that observed for the A site. Modeling of the aminoglycoside-DIS complex allowed us to identify antibiotic modifications likely to increase the affinity and/or the specificity for the DIS. This could be a starting point for designing antiviral drugs against HIV-1 RNA dimerization.The fight against HIV 1 relies essentially on multitherapies targeting two viral enzymes: reverse transcriptase and protease. Due to a highly variable genome leading to rapid selection of mutations that confer resistance to enzymatic inhibitors, there is a strong need for targeting new viral molecules. New potential targets such as viral and cellular proteins involved in viral adsorption, fusion, and integration have been proposed (1). Functional sites within the genomic RNA, such as the Tat-responsive element (2) and the Rev-responsive element (3), have also been described as potential targets for the aminoglycoside antibiotic neomycin B.Another interesting viral RNA target is the dimerization initiation site (DIS) of HIV-1 genomic RNA. This stem-loop is highly conserved among the different HIV-1 subtypes identified by sequence alignment. It initiates dimerization by forming a kissing-loop complex through base-pairing of a 6-nucleotide self-complementary sequence in each loop (4 -8) (Fig. 1). It has been shown that this kissing-loop complex is converted in vitro by the nucleocapsid protein into a more stable complex, assumed to correspond to an extended duplex (9). Stabilization of the RNA dimer was also observed during maturation of the viral particles (10). Genome dimerization facilitates recombination (11) and is required for efficient RNA packaging (12-15) and reverse transcription (13, 16). Alteration of the DIS dramatically reduces viral infectivity (12-15).We recently solved the crystal structures of the DIS kissingloop complexes of HIV-1 subtypes A and B (17). In the present study, we show that, unexpectedly, these structures are very similar to the ribosomal aminoacyl decoding site (A site) complexed with paromomycin (18, 19), an aminoglycoside antibiotic interfering with translation in bacteria. Based on this similarity, we modeled a DIS kissing-loop complex binding two paromomycin molecules. This model is...

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“…The vast majority of the information on aminoglycoside interactions with their respective RNA targets has come from the analysis of drug binding to model RNA substrates (for example, Recht et al 1996;Vicens and Westhof 2001;Llano-Sotelo et al 2002;Vicens and Westhof 2002;Pilch et al 2003;Francois et al 2005). Although the studies of drug-RNA interactions based on the use of short RNA constructs were illuminating (Recht et al 1996;Lynch et al 2003), the crystal structures of ribosomal subunit complexes show that more dramatic conformational changes are observed in the rRNA of the intact 30S subunit upon aminoglycoside binding compared to those of the simplified substrates Lynch et al 2003). Furthermore, only studies involving the entire ribosomal subunit are capable of revealing the effects of global conformational changes and of other ribosomal ligands on antibiotic binding, which underscores the utility of the technique described here.…”

Section: Discussionmentioning

confidence: 99%

Fluorescently labeled ribosomes as a tool for analyzing antibiotic binding

Llano-Sotelo¹,

Hickerson²,

Lancaster³

et al. 2009

RNA

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Measuring the binding of antibiotics and other small-molecular-weight ligands to the 2.5 MDa ribosome often presents formidable challenges. Here, we describe a general method for studying binding of ligands to ribosomes that carry a site-specific fluorescent label covalently attached to one of the ribosomal proteins. As a proof of principle, an environment-sensitive fluorescent group was placed at several specific sites within the ribosomal protein S12. Small ribosomal subunits were reconstituted from native 16S rRNA, individually purified small subunit proteins, and fluorescently labeled S12. The fluorescence characteristics of the reconstituted subunits were affected by several antibiotics, including streptomycin and neomycin, which bind in the vicinity of protein S12. The equilibrium dissociation constants of the drugs obtained using a conventional fluorometer were in good agreement with those observed using previously published methods and with measurements based on the use of radiolabeled streptomycin. The newly developed method is rapid and sensitive, and can be used for determining thermodynamic and kinetic binding characteristics of antibiotics and other small ribosomal ligands. The method can readily be adapted for use in high-throughput screening assays.

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RNA Sequence Determinants for Aminoglycoside Binding to an A-site rRNA Model Oligonucleotide

Cited by 246 publications

References 43 publications

Plasmid-Mediated High-Level Resistance to Aminoglycosides in Enterobacteriaceae Due to 16S rRNA Methylation

Plasmid-Mediated High-Level Resistance to Aminoglycosides in Enterobacteriaceae Due to 16S rRNA Methylation

HIV-1 RNA Dimerization Initiation Site Is Structurally Similar to the Ribosomal A Site and Binds Aminoglycoside Antibiotics

Fluorescently labeled ribosomes as a tool for analyzing antibiotic binding

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