PMID:12930958
| Citation |
Xaplanteri, MA, Andreou, A, Dinos, GP and Kalpaxis, DL (2003) Effect of polyamines on the inhibition of peptidyltransferase by antibiotics: revisiting the mechanism of chloramphenicol action. Nucleic Acids Res. 31:5074-83 |
|---|---|
| Abstract |
Chloramphenicol is thought to interfere competitively with the binding of the aminoacyl-tRNA 3'-terminus to ribosomal A-site. However, noncompetitive or mixed-noncompetitive inhibition, often observed to be dependent on chloramphenicol concentration and ionic conditions, leaves some doubt about the precise mode of action. Here, we examine further the inhibition effect of chloramphenicol, using a model system derived from Escherichia coli in which a peptide bond is formed between puromycin and AcPhe-tRNA bound at the P-site of poly(U)-programmed ribosomes, under ionic conditions (6 mM Mg2+, 100 mM NH4+, 100 microM spermine) more closely resembling the physiological status. Kinetics reveal that chloramphenicol (I) reacts rapidly with AcPhe-tRNA.poly(U).70S ribosomal complex (C) to form the encounter complex CI which is then isomerized slowly to a more tight complex, C*I. A similar inhibition pattern is observed, if complex C modified by a photoreactive analogue of spermine, reacts in buffer free of spermine. Spermine, either reversibly interacting with or covalently attached to ribosomes, enhances the peptidyltransferase activity and increases the chloramphenicol potency, without affecting the isomerization step. As indicated by photoaffinity labeling, the peptidyltransferase center at which chloramphenicol binds, is one of the preferred cross-linking sites for polyamines. This fact may explain the effect of spermine on chloramphenicol binding to ribosomes. |
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| Keywords |
Anti-Bacterial Agents/metabolism; Anti-Bacterial Agents/pharmacology; Azides/metabolism; Azides/pharmacology; Base Sequence; Binding Sites; Binding, Competitive/drug effects; Chloramphenicol/metabolism; Chloramphenicol/pharmacology; Dose-Response Relationship, Drug; Escherichia coli/genetics; Escherichia coli/metabolism; Kinetics; Magnesium/pharmacology; Molecular Sequence Data; Nucleic Acid Conformation; Peptide Biosynthesis/drug effects; Peptidyl Transferases/antagonists & inhibitors; Peptidyl Transferases/metabolism; Polyamines/metabolism; Polyamines/pharmacology; Puromycin/metabolism; RNA, Ribosomal, 23S/chemistry; RNA, Ribosomal, 23S/genetics; RNA, Ribosomal, 23S/metabolism; RNA, Transfer, Amino Acyl/metabolism; Ribosomes/metabolism; Spermine/analogs & derivatives; Spermine/metabolism; Spermine/pharmacology |
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