Protected nucleotide G2608 in 23S rRNA confers resistance to oxazolidinones in E. coli

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Abstract

The oxazolidinones are a new class of potent antibiotics that are active against a broad spectrum of Gram-positive bacterial pathogens including those resistant to other antibiotics. These drugs specifically inhibit protein biosynthesis whereas DNA and RNA synthesis are not affected. Although biochemical and genetic studies indicate that oxazolidinones target the ribosomal peptidyltransferase center, other investigations suggest that they interact with different regions of ribosomes. Thus, the exact binding site and mechanism of action have remained elusive. Here, we study, by use of base-specific reagents, the effect of the oxazolidinones on the chemical protection footprinting patterns of the 23S rRNA. We report: (i) reproducible protection of G2607 and G2608 of 23S rRNA by a potent oxazolidinone on a ribosome·tRNA·mRNA complex; (ii) no protections were observed on 70S ribosomes devoid of tRNA and mRNA; (iii) EF-G also weakly protected G2607 and G2608; (iv) mutations at G2608 conferred resistance to the oxazolidinones in Escherichia coli cells; and (v) G2607 and G2608 occur near the exit to the peptide tunnel on the 50S subunit. A mechanism for the pleiotropic action of the oxazolidinones is discussed.

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Materials

Dimethyl sulfate (DMS), diethyl pyrocarbonate (DEP), kethoxal, carbodiimide (CMCT), poly(U), tRNAPhe, fusidic acid, thiostrepton, lincomycin, chloramphenicol, and tetracycline antibiotics were from Sigma. [α-35S]dATP (>1000 Ci/mmol) was obtained from Amersham. DNA and RNA were purified using Qiagen kits. The sequencing reactions were performed with the USB sequencing kit. The oxazolidinone III was from Pharmacia (Pfizer) and was described before [13]. Escherichia coli strain HN818, which is

Chemical protection footprinting of the oxazolidinone III on ribosomes

Controversial results about the oxazolidione binding site have been reported possibly due to the low binding affinity of oxazolidinones for the isolated ribosomes. Using UV-induced cross-linking and footprinting, the binding sites were determined to be A864 in the 16S rRNA and several nucleotides in the vicinity of ribosomal E-site in 23S rRNA [8]. However, more recent evidence indicates that oxazolidinones interact with the 50S ribosomal subunit [7], [26]. Our previous results revealed that

Acknowledgments

We are grateful to the Pharmacia/Upjohn Corporation (Pfizer) and to the Canadian NSERC for financial support. J. Xu received a post-doctoral fellowship from the CIHR, which is gratefully acknowledged. We thank Dr. Hiroshi Nikaido at University of California at Berkeley for providing the oxazolidinone-sensitive E. coli strain HN818. This work is dedicated to the memory of Clelia H. Finney.

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