Tetracycline does not directly inhibit the function of bacterial elongation factor Tu
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 gene...
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| description | 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 estimated 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-based kinetic assay that minimizes the effect of tetracycline autofluorescence, enabling the detailed kinetic analysis of the nucleotide-binding properties of Escherichia coli EF-Tu. Furthermore, using physiologically 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-tRNAPhe complex. We therefore provide clear evidence that tetracycline does not directly impede the function of EF-Tu. |
| doi_str_mv | 10.1371/journal.pone.0178523 |
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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 estimated 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-based kinetic assay that minimizes the effect of tetracycline autofluorescence, enabling the detailed kinetic analysis of the nucleotide-binding properties of Escherichia coli EF-Tu. Furthermore, using physiologically 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-tRNAPhe complex. 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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 estimated 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-based kinetic assay that minimizes the effect of tetracycline autofluorescence, enabling the detailed kinetic analysis of the nucleotide-binding properties of Escherichia coli EF-Tu. Furthermore, using physiologically 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-tRNAPhe complex. We therefore provide clear evidence that tetracycline does not directly impede the function of EF-Tu.</description><subject>Alignment</subject><subject>Amino acid sequence</subject><subject>Amino acids</subject><subject>Animal health</subject><subject>Antibiotic resistance</subject><subject>Antibiotics</subject><subject>Assaying</subject><subject>Bacteria</subject><subject>Bacteria - metabolism</subject><subject>Binding sites</subject><subject>Biochemistry</subject><subject>Biology and Life Sciences</subject><subject>Bonding</subject><subject>Chemical bonds</subject><subject>Classification</subject><subject>Communication networks</subject><subject>Conserved sequence</subject><subject>Construction</subject><subject>Cracks</subject><subject>Crystal structure</subject><subject>Crystallography</subject><subject>Cysteine</subject><subject>Decoding</subject><subject>E coli</subject><subject>Elongation</subject><subject>Elongation factor EF-Tu</subject><subject>Escherichia coli</subject><subject>Fluorescence</subject><subject>Genetic aspects</subject><subject>Gingiva</subject><subject>GTP</subject><subject>GTPases</subject><subject>Guanine</subject><subject>Guanosine</subject><subject>Guanosine triphosphatases</subject><subject>Guanosine triphosphate</subject><subject>Guanosinetriphosphatase</subject><subject>Gums</subject><subject>Histidine</subject><subject>Hydrolysis</subject><subject>Inhibitors</subject><subject>Kinetics</subject><subject>Libraries</subject><subject>Magnesium</subject><subject>Medicine and Health Sciences</subject><subject>Minimum inhibitory concentration</subject><subject>Molecular modelling</subject><subject>Molecular structure</subject><subject>Nucleic acids</subject><subject>Nucleotide sequence</subject><subject>Nucleotides</subject><subject>Pathogens</subject><subject>Peptide Elongation Factor Tu - antagonists & inhibitors</subject><subject>Phosphates</subject><subject>Physical Sciences</subject><subject>Physiological aspects</subject><subject>Proteins</subject><subject>Residues</subject><subject>Ribonucleic acid</subject><subject>RNA</subject><subject>Stacking</subject><subject>Switching theory</subject><subject>Tetracycline - pharmacology</subject><subject>Tetracyclines</subject><subject>Training</subject><issn>1932-6203</issn><issn>1932-6203</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2017</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><sourceid>BENPR</sourceid><sourceid>DOA</sourceid><recordid>eNqNkl1r2zAUhs3YWLtu_2BshsHYLpLpy7J9MyhlH4FCYUt3KyRZShQUKZXksfz7yYlb4tGLoQuLc57zynr1FsVrCOYQ1_DTxvfBcTvfeafmANZNhfCT4hy2GM0oAvjpyf6seBHjBoAKN5Q-L85QU1WobeB5cbtUKXC5l9Y4VXZexdL5VHYmKJnsvjRubYRJZVqrUvdOJuNd6XUpuEwqGG5LZb1b8UNd56IP5bJ_WTzT3Eb1avxeFLdfvyyvvs-ub74tri6vZ5K2KM0QrTGnFaWt4LVqKRANJ1pD1IGOdF0tZU0lkAhTLjABUIhK5YZopJYNohhfFG-PujvrIxsdiQy2ANGqIbDJxOJIdJ5v2C6YLQ975rlhh4IPK8ZDMtIqhiUiUirOdUUI7pTQtIYdkEQMDQ6z1ufxtF5sVSeVy9bZiei048yarfxvlvUorGkW-DAKBH_Xq5jY1kSprOVO-f7w35gQACjJ6Lt_0MdvN1Irni9gnPbDYw6i7JK0qCU1qgaX5o9QeXVqa2SOjza5Phn4OBnITFJ_0or3MbLFzx__z978mrLvT9i14jato7f9EJ44BckRlMHHGJR-MBkCNqT_3g02pJ-N6c9jb04f6GHoPu74L-j3AK8</recordid><startdate>20170526</startdate><enddate>20170526</enddate><creator>Gzyl, Katherine E</creator><creator>Wieden, Hans-Joachim</creator><general>Public Library of Science</general><general>Public Library of Science (PLoS)</general><scope>CGR</scope><scope>CUY</scope><scope>CVF</scope><scope>ECM</scope><scope>EIF</scope><scope>NPM</scope><scope>AAYXX</scope><scope>CITATION</scope><scope>IOV</scope><scope>ISR</scope><scope>3V.</scope><scope>7QG</scope><scope>7QL</scope><scope>7QO</scope><scope>7RV</scope><scope>7SN</scope><scope>7SS</scope><scope>7T5</scope><scope>7TG</scope><scope>7TM</scope><scope>7U9</scope><scope>7X2</scope><scope>7X7</scope><scope>7XB</scope><scope>88E</scope><scope>8AO</scope><scope>8C1</scope><scope>8FD</scope><scope>8FE</scope><scope>8FG</scope><scope>8FH</scope><scope>8FI</scope><scope>8FJ</scope><scope>8FK</scope><scope>ABJCF</scope><scope>ABUWG</scope><scope>AEUYN</scope><scope>AFKRA</scope><scope>ARAPS</scope><scope>ATCPS</scope><scope>AZQEC</scope><scope>BBNVY</scope><scope>BENPR</scope><scope>BGLVJ</scope><scope>BHPHI</scope><scope>C1K</scope><scope>CCPQU</scope><scope>D1I</scope><scope>DWQXO</scope><scope>FR3</scope><scope>FYUFA</scope><scope>GHDGH</scope><scope>GNUQQ</scope><scope>H94</scope><scope>HCIFZ</scope><scope>K9.</scope><scope>KB.</scope><scope>KB0</scope><scope>KL.</scope><scope>L6V</scope><scope>LK8</scope><scope>M0K</scope><scope>M0S</scope><scope>M1P</scope><scope>M7N</scope><scope>M7P</scope><scope>M7S</scope><scope>NAPCQ</scope><scope>P5Z</scope><scope>P62</scope><scope>P64</scope><scope>PATMY</scope><scope>PDBOC</scope><scope>PIMPY</scope><scope>PQEST</scope><scope>PQQKQ</scope><scope>PQUKI</scope><scope>PRINS</scope><scope>PTHSS</scope><scope>PYCSY</scope><scope>RC3</scope><scope>7X8</scope><scope>5PM</scope><scope>DOA</scope><orcidid>https://orcid.org/0000-0001-5181-6059</orcidid></search><sort><creationdate>20170526</creationdate><title>Tetracycline does not directly inhibit the function of bacterial elongation factor Tu</title><author>Gzyl, Katherine E ; Wieden, Hans-Joachim</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c692t-2673a65669ba7e960b8a4ff12d0d4dd7cc76c0c236ab3401bb5ed4db8cfc82633</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2017</creationdate><topic>Alignment</topic><topic>Amino acid sequence</topic><topic>Amino acids</topic><topic>Animal health</topic><topic>Antibiotic resistance</topic><topic>Antibiotics</topic><topic>Assaying</topic><topic>Bacteria</topic><topic>Bacteria - metabolism</topic><topic>Binding sites</topic><topic>Biochemistry</topic><topic>Biology and Life Sciences</topic><topic>Bonding</topic><topic>Chemical bonds</topic><topic>Classification</topic><topic>Communication networks</topic><topic>Conserved sequence</topic><topic>Construction</topic><topic>Cracks</topic><topic>Crystal structure</topic><topic>Crystallography</topic><topic>Cysteine</topic><topic>Decoding</topic><topic>E coli</topic><topic>Elongation</topic><topic>Elongation factor EF-Tu</topic><topic>Escherichia coli</topic><topic>Fluorescence</topic><topic>Genetic aspects</topic><topic>Gingiva</topic><topic>GTP</topic><topic>GTPases</topic><topic>Guanine</topic><topic>Guanosine</topic><topic>Guanosine triphosphatases</topic><topic>Guanosine triphosphate</topic><topic>Guanosinetriphosphatase</topic><topic>Gums</topic><topic>Histidine</topic><topic>Hydrolysis</topic><topic>Inhibitors</topic><topic>Kinetics</topic><topic>Libraries</topic><topic>Magnesium</topic><topic>Medicine and Health Sciences</topic><topic>Minimum inhibitory concentration</topic><topic>Molecular modelling</topic><topic>Molecular structure</topic><topic>Nucleic acids</topic><topic>Nucleotide sequence</topic><topic>Nucleotides</topic><topic>Pathogens</topic><topic>Peptide Elongation Factor Tu - antagonists & inhibitors</topic><topic>Phosphates</topic><topic>Physical Sciences</topic><topic>Physiological aspects</topic><topic>Proteins</topic><topic>Residues</topic><topic>Ribonucleic acid</topic><topic>RNA</topic><topic>Stacking</topic><topic>Switching theory</topic><topic>Tetracycline - pharmacology</topic><topic>Tetracyclines</topic><topic>Training</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Gzyl, Katherine E</creatorcontrib><creatorcontrib>Wieden, Hans-Joachim</creatorcontrib><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>Gale In Context: Opposing Viewpoints</collection><collection>Gale In Context: Science</collection><collection>ProQuest Central (Corporate)</collection><collection>Animal Behavior Abstracts</collection><collection>Bacteriology Abstracts (Microbiology B)</collection><collection>Biotechnology Research Abstracts</collection><collection>Nursing & Allied Health Database</collection><collection>Ecology Abstracts</collection><collection>Entomology Abstracts (Full archive)</collection><collection>Immunology Abstracts</collection><collection>Meteorological & Geoastrophysical Abstracts</collection><collection>Nucleic Acids Abstracts</collection><collection>Virology and AIDS Abstracts</collection><collection>Agricultural Science Collection</collection><collection>Health & Medical Collection</collection><collection>ProQuest Central (purchase pre-March 2016)</collection><collection>Medical Database (Alumni Edition)</collection><collection>ProQuest Pharma Collection</collection><collection>Public Health Database</collection><collection>Technology Research Database</collection><collection>ProQuest SciTech Collection</collection><collection>ProQuest Technology Collection</collection><collection>ProQuest Natural Science Collection</collection><collection>Hospital Premium Collection</collection><collection>Hospital Premium Collection (Alumni Edition)</collection><collection>ProQuest Central (Alumni) (purchase pre-March 2016)</collection><collection>Materials Science & Engineering Collection</collection><collection>ProQuest Central (Alumni Edition)</collection><collection>ProQuest One Sustainability</collection><collection>ProQuest Central UK/Ireland</collection><collection>Advanced Technologies & Aerospace Collection</collection><collection>Agricultural & Environmental Science Collection</collection><collection>ProQuest Central Essentials</collection><collection>Biological Science Collection</collection><collection>ProQuest Central</collection><collection>Technology Collection</collection><collection>Natural Science Collection</collection><collection>Environmental Sciences and Pollution Management</collection><collection>ProQuest One Community College</collection><collection>ProQuest Materials Science Collection</collection><collection>ProQuest Central Korea</collection><collection>Engineering Research Database</collection><collection>Health Research Premium Collection</collection><collection>Health Research Premium Collection (Alumni)</collection><collection>ProQuest Central Student</collection><collection>AIDS and Cancer Research Abstracts</collection><collection>SciTech Premium Collection</collection><collection>ProQuest Health & Medical Complete (Alumni)</collection><collection>Materials Science Database</collection><collection>Nursing & Allied Health Database (Alumni Edition)</collection><collection>Meteorological & Geoastrophysical Abstracts - 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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 estimated 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-based kinetic assay that minimizes the effect of tetracycline autofluorescence, enabling the detailed kinetic analysis of the nucleotide-binding properties of Escherichia coli EF-Tu. Furthermore, using physiologically 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-tRNAPhe complex. We therefore provide clear evidence that tetracycline does not directly impede the function of EF-Tu.</abstract><cop>United States</cop><pub>Public Library of Science</pub><pmid>28552981</pmid><doi>10.1371/journal.pone.0178523</doi><tpages>e0178523</tpages><orcidid>https://orcid.org/0000-0001-5181-6059</orcidid><oa>free_for_read</oa></addata></record> |
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| subjects | Alignment Amino acid sequence Amino acids Animal health Antibiotic resistance Antibiotics Assaying Bacteria Bacteria - metabolism Binding sites Biochemistry Biology and Life Sciences Bonding Chemical bonds Classification Communication networks Conserved sequence Construction Cracks Crystal structure Crystallography Cysteine Decoding E coli Elongation Elongation factor EF-Tu Escherichia coli Fluorescence Genetic aspects Gingiva GTP GTPases Guanine Guanosine Guanosine triphosphatases Guanosine triphosphate Guanosinetriphosphatase Gums Histidine Hydrolysis Inhibitors Kinetics Libraries Magnesium Medicine and Health Sciences Minimum inhibitory concentration Molecular modelling Molecular structure Nucleic acids Nucleotide sequence Nucleotides Pathogens Peptide Elongation Factor Tu - antagonists & inhibitors Phosphates Physical Sciences Physiological aspects Proteins Residues Ribonucleic acid RNA Stacking Switching theory Tetracycline - pharmacology Tetracyclines Training |
| title | Tetracycline does not directly inhibit the function of bacterial elongation factor Tu |
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