Binding of neomycin-class aminoglycoside antibiotics to the A-site of 16 s rRNA

Fourmy, Dominique; Recht, Michael I.; Puglisi, Joseph D.

doi:10.1006/jmbi.1997.1552

Cited by 242 publications

(224 citation statements)

References 23 publications

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“…9 The binding of GN to (E‚S)o at low pH is stronger than at high pH over the entire range of Mg 2+ and Mn 2+ concentrations investigated [ K b G N g 60 mM -1 at pH 4.7-5.0 ( Figure 8); K b G N e 9.1 mM -1 above pH 7.9 (1 and unpublished results)]; this suggests that G NH 3 + binds more strongly to the (E‚S)o complex than GNH 2 . The binding of GN to (E‚S)o observed below pH 5.0 can therefore be attributed to G NH 3 +, with negligible contribution from the small fraction of GNH 2 that is present (e0.04; pK a G N ) 6.1).…”

Section: The Binding Of G Nhmentioning

confidence: 99%

“…The backbone of RNA is composed of negatively charged phosphoryl groups; this results in repulsive interactions, but also creates numerous potential binding sites for metal ions and other positively charged groups (e.g., 2-15 and references cited therein). The 2′-hydroxyl groups and functional groups on the bases of RNA also provide hydrogen bond donors and acceptors that can interact with each other, with metal ions, and with polar groups on bound ligands (e.g., 3,4,6,8,9,[11][12][13][14][16][17][18][19][20][21], and references cited therein). The ring moieties of the bases are nonpolar, creating the potential for hydrophobic interactions with each other and with bound ligands (e.g., 19, 21, and references cited therein).…”

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confidence: 99%

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Protonated 2‘-Aminoguanosine as a Probe of the Electrostatic Environment of the Active Site of the Tetrahymena Group I Ribozyme

1999

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We have probed the electrostatic environment of the active site of the Tetrahymena group I ribozyme (E) using protonated 2′-aminoguanosine (G NH 3 +), in which the 2′-OH of the guanosine nucleophile (G) is replaced by an -NH 3 + group. At low concentrations of divalent metal ion (2 mM Mg 2+ ), G NH 3 + binds at least 200-fold stronger than G or G NH 2 , with a dissociation constant of e1 µM from the ribozyme‚oligonucleotide substrate‚G NH 3 + complex (E‚S‚G NH 3 +). This strong binding suggests that the -NH 3 + group interacts with negatively charged phosphoryl groups within the active site. Increasing the concentration of divalent metal ion weakens the binding of G NH 3 + to E‚S more than 10 2 -fold. The Mn 2+ concentration dependence suggests that M C , the metal ion that interacts with the 2′-moiety of G in the normal reaction, is responsible for this effect. M C and G NH 3 + compete for binding to the active site; this competition could arise from electrostatic repulsion between the positively charged -NH 3 + and M C and, possibly, from their competition for interaction with active site phosphoryl groups. The reactive phosphoryl group of S increases the competition between M C and G NH 3 +, consistent with a network of interactions involving M C that help position the reactive phosphoryl group and the guanosine nucleophile with respect to one another. The chemical step with bound G NH 3 + is at least 10 4 -fold slower than with G or G NH 2 . These results provide additional support for an integral role of M C in catalysis by the Tetrahymena ribozyme, and demonstrate the utility of the -NH 3 + moiety as an electrostatic probe within a structured RNA.An RNA enzyme can utilize a variety of functional groups that differ in charge and polarity for building its active site and interacting with its substrates. The backbone of RNA is composed of negatively charged phosphoryl groups; this results in repulsive interactions, but also creates numerous potential binding sites for metal ions and other positively charged groups (e.g., 2-15 and references cited therein). The 2′-hydroxyl groups and functional groups on the bases of RNA also provide hydrogen bond donors and acceptors that can interact with each other, with metal ions, and with polar groups on bound ligands (e.g., 3, 4, 6, 8, 9, 11-14, 16-21, and references cited therein). The ring moieties of the bases are nonpolar, creating the potential for hydrophobic interactions with each other and with bound ligands (e.g., 19, 21, and references cited therein).The energetic effects of electrostatic interactions between functional groups within an RNA also depend on the ability of the RNA to position these groups and to limit active site rearrangements. The Tetrahymena group I ribozyme makes extensive interactions with its substrates, which has been suggested to allow precise positioning of substrates and catalytic groups within this RNA active site (22-25 and references cited therein). The interactions within the ribozyme core presumably form an interconnecte...

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Section: The Binding Of G Nhmentioning

confidence: 99%

mentioning

confidence: 99%

Protonated 2‘-Aminoguanosine as a Probe of the Electrostatic Environment of the Active Site of the Tetrahymena Group I Ribozyme

1999

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“…Recent studies suggested that the minimal motif for specific binding of aminoglycosides to the A site of the ribosome is ring I and ring II (neamine core). 15 In fact, several analogues based on the neamine core have comparable or better binding affinities and antimicrobial activities than the parent aminoglycosides. 19,20 Aminoglycosides interact directly with the conserved regions of ribosomal RNA.…”

Section: Introductionmentioning

confidence: 99%

Monitoring aminoglycoside-induced conformational changes in 16S rRNA through acrylamide quenching

Chao

Chow

2007

Bioorganic & Medicinal Chemistry

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Fluorescence of 2-aminopurine (2AP) substituted A site and acrylamide quenching were used to study the interactions of paromomycin and neamine with the decoding region of 16S rRNA. The results reveal that paromomycin binding to the A-site RNA leads to increased exposure of residue A1492. In contrast, neamine has little effect on the solvent accessibility of A1492. Electrospray ionization mass spectrometry was used to compare the affinity of paromomycin with the A-site and 2-AP-substituted A-site RNAs.

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“…As previously observed with other RNA ligands, a relative weak EC 50 (around 10 mM) does not imply poor inhibitor activity, as shown for TAR-Argininamide or neomycin-16S rRNA complexes. 60,61 Although the inhibition induced by the IRAB benzimidazole compound is detected at a concentration of 1 to 2 mM in in vitro translation assays, the conformational changes induced on the RNA structure of the full-length IRES in the context of a functional mRNA were detectable at 10-fold higher concentration (20 mM) by SHAPE methodology. Taken together, these results indicate that subtle structural changes of the IRES conformation could lead to drastic effects over its activity.…”

Section: Discussionmentioning

confidence: 99%

Local RNA flexibility perturbation of the IRES element induced by a novel ligand inhibits viral RNA translation

et al. 2015

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(2015) Local RNA flexibility perturbation of the IRES element induced by a novel ligand inhibits viral RNA translation, RNA Biology, 12:5, 555-568, DOI: 10.1080/15476286.2015 The internal ribosome entry site (IRES) element located at the 5´untranslated genomic region of various RNA viruses mediates cap-independent initiation of translation. Picornavirus IRES activity is highly dependent on both its structural organization and its interaction with host factors. Small molecules able to interfere with RNA function are valuable candidates for antiviral agents. Here we show that a small molecule based on benzimidazole (IRAB) inhibited foot-andmouth disease virus (FMDV) IRES-dependent protein synthesis in cells transfected with infectious RNA leading to a decrease of the virus titer, which was higher than that induced by a structurally related benzimidazole derivative. Interestingly, IRAB preferentially inhibited IRES-dependent translation in cell free systems in a dose-dependent manner. RNA structural analysis by SHAPE demonstrated an increased local flexibility of the IRES structure upon incubation with IRAB, which affected 3 stem-loops (SL) of domain 3. Fluorescence binding assays conducted with individual aminopurinelabeled oligoribonucleotides indicated that the SL3A binds IRAB (EC 50 18 mM). Taken together, the results derived from SHAPE reactivity and fluorescence binding assays suggested that the target site of IRAB within the FMDV IRES might be a folded RNA structure that involves the entire apical region of domain 3. Our data suggest that the conformational changes induced by this compound on a specific region of the IRES structure which is essential for its activity is, at least in part, responsible for the reduced IRES efficiency observed in cell free lysates and, particularly, in RNA-transfected cells.

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Binding of neomycin-class aminoglycoside antibiotics to the A-site of 16 s rRNA

Cited by 242 publications

References 23 publications

Protonated 2‘-Aminoguanosine as a Probe of the Electrostatic Environment of the Active Site of the Tetrahymena Group I Ribozyme

Protonated 2‘-Aminoguanosine as a Probe of the Electrostatic Environment of the Active Site of the Tetrahymena Group I Ribozyme

Monitoring aminoglycoside-induced conformational changes in 16S rRNA through acrylamide quenching

Local RNA flexibility perturbation of the IRES element induced by a novel ligand inhibits viral RNA translation

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