Biochemical and Biophysical Research Communications
Protected nucleotide G2608 in 23S rRNA confers resistance to oxazolidinones in E. coli
Section snippets
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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