A conserved secondary structural motif in 23S rRNA defines the site of interaction of amicetin, a universal inhibitor of peptide bond formation.

Abstract

The binding site and probable site of action have been determined for the universal antibiotic amicetin which inhibits peptide bond formation. Evidence from in vivo mutants, site‐directed mutations and chemical footprinting all implicate a highly conserved motif in the secondary structure of the 23S‐like rRNA close to the central circle of domain V. We infer that this motif lies at, or close to, the catalytic site in the peptidyl transfer centre. The binding site of amicetin is the first of a group of functionally related hexose‐cytosine inhibitors to be localized on the ribosome.

NMR and Molecular Modelling Studies of the Binding of Amicetin Antibiotic to Conserved Secondary Structural Motifs of 23S Ribosomal RNAs

“…1a), for amicetin binding based on the results previously reported in the literature [12] and discussed above. The underlined nucleotides of the hexanucleotide linker (5Ј-CUUCGG-3Ј), occuring commonly in ribosomal RNAs, has been shown to form a stable tetraloop and confer high thermal stability [23].…”

“…1a) [4,5]. However, based on more recent experiments, it has been revealed that antibiotic inhibition of peptide transfer can also take place by other mechanisms, attributing functional and structural roles for analogous conserved structural motifs lying adjacent to the catalytic transfer centre [1,12]. One such antibiotic, amicetin, belongs to a functionally related family of drugs which share a hexosecytosine moiety (Fig.…”

“…1b) [2,13]. Amicetin has been described as an 'universal' antibiotic as it binds to a highly conserved structural motif of 23S like rRNAs which occur in organisms of different evolutionary origins (archae, bacteria and eukarya) [12]. The secondary structures of the highly conserved rRNA motifs of Hh and E. coli within the central circle domain V, with affinity for amicetin, are illustrated in Figure 1c, d.…”

“…It has been postulated that whereas some drugs belonging to the category of chloramphenicol and macrolide antibiotics may impair initial binding of the aminoacyl tRNA in the A-site, other hexose-cytosine drugs such as amicetin may block the movement of the aminoacyl group towards the P-site bound peptidyl tRNA and that the drug binding sites lie very close to the catalytic centre [4,12]. Previously, amicetin binding to 23S rRNA of intact Hh ribosomes were probed by chemical footprinting experiments using nucleotide specific chemical modification reagents in order to establish the amicetin binding site [12]. However, these experiments failed to yield discernible footprints and hence exact details regarding the site and mode of amicetin binding to Hh rRNA have remained obscure, despite the generation of a spontaneous single nucleotide amicetin-resistant mutant (U2457→C) strongly implying amicetin binding [12].…”

“…Previously, amicetin binding to 23S rRNA of intact Hh ribosomes were probed by chemical footprinting experiments using nucleotide specific chemical modification reagents in order to establish the amicetin binding site [12]. However, these experiments failed to yield discernible footprints and hence exact details regarding the site and mode of amicetin binding to Hh rRNA have remained obscure, despite the generation of a spontaneous single nucleotide amicetin-resistant mutant (U2457→C) strongly implying amicetin binding [12]. Similar "non-footprinting" antibiotics such as sparsomycin (most potent inhibitor of protein synthesis ) involved in ribosomal activity, causing a specific resistance mutation of Hh 23S rRNA have been reported [14].…”