Wieden, Hans-Joachim

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Browsing Wieden, Hans-Joachim by Subject "Anti-infective agents"

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    Tetracycline does not directly inhibit the function of bacterial elongation factor Tu
    (Public Library of Science, 2017) Gzyl, Katherine E.; Wieden, Hans-Joachim
    Understanding the molecular mechanism of antibiotics that are currently in use is important for the development of new antimicrobials. The tetracyclines, discovered in the 1940s, are a well-established class of antibiotics that still have a role in treating microbial infections in humans. It is generally accepted that the main target of their action is the ribosome. The esti- mated affinity for tetracycline binding to the ribosome is relatively low compared to the actual potency of the drug in vivo . Therefore, additional inhibitory effects of tetracycline on the translation machinery have been discussed. Structural evidence suggests that tetracycline inhibits the function of the essential bacterial GTPase Elongation Factor (EF)-Tu through interaction with the bound nucleotide. Based on this, tetracycline has been predicted to impede the nucleotide-binding properties of EF-Tu. However, detailed kinetic studies addressing the effect of tetracycline on nucleotide binding have been prevented by the fluorescence properties of the antibiotic. Here, we report a fluorescence-bas ed kinetic assay that minimizes the effect of tetracycline autofluorescence, enabling the detailed kinetic analysis of the nucleotide-bin ding properties of Escherichia coli EF-Tu. Further- more, using physiologica lly relevant conditions, we demonstrate that tetracycline does not affect EF-Tu’s intrinsic or ribosome-stimulated GTPase activity, nor the stability of the EF- Tu•GTP•Phe-tRNA Phe complex. We therefore provide clear evidence that tetracycline does not directly impede the function of EF-Tu.