Novel Escherichia coli RF1 mutants with decreased translation termination activity and increased sensitivity to the cytotoxic effect of the bacterial toxins Kid and RelE
Novel mutations in prfA, the gene for the polypeptide release factor RF1 of Escherichia coli, were isolated using a positive genetic screen based on the parD (kis, kid) toxin-antitoxin system. This original approach allowed the direct selection of mutants with altered translational termination effic...
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| Veröffentlicht in: | Molecular microbiology 2009-01, Vol.71 (1), p.66-78 |
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| creator | Diago-Navarro, Elizabeth Mora, Liliana Buckingham, Richard H Díaz-Orejas, Ramón Lemonnier, Marc |
| description | Novel mutations in prfA, the gene for the polypeptide release factor RF1 of Escherichia coli, were isolated using a positive genetic screen based on the parD (kis, kid) toxin-antitoxin system. This original approach allowed the direct selection of mutants with altered translational termination efficiency at UAG codons. The isolated prfA mutants displayed a ~10-fold decrease in UAG termination efficiency with no significant changes in RF1 stability in vivo. All three mutations, G121S, G301S and R303H, were situated close to the nonsense codon recognition site in RF1:ribosome complexes. The prfA mutants displayed increased sensitivity to the RelE toxin encoded by the relBE system of E. coli, thus providing in vivo support for the functional interaction between RF1 and RelE. The prfA mutants also showed increased sensitivity to the Kid toxin. Since this toxin can cleave RNA in a ribosome-independent manner, this result was not anticipated and provided first evidence for the involvement of RF1 in the pathway of Kid toxicity. The sensitivity of the prfA mutants to RelE and Kid was restored to normal levels upon overproduction of the wild-type RF1 protein. We discuss these results and their utility for the design of novel antibacterial strategies in the light of the recently reported structure of ribosome-bound RF1. |
| doi_str_mv | 10.1111/j.1365-2958.2008.06510.x |
| format | Article |
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This original approach allowed the direct selection of mutants with altered translational termination efficiency at UAG codons. The isolated prfA mutants displayed a ~10-fold decrease in UAG termination efficiency with no significant changes in RF1 stability in vivo. All three mutations, G121S, G301S and R303H, were situated close to the nonsense codon recognition site in RF1:ribosome complexes. The prfA mutants displayed increased sensitivity to the RelE toxin encoded by the relBE system of E. coli, thus providing in vivo support for the functional interaction between RF1 and RelE. The prfA mutants also showed increased sensitivity to the Kid toxin. Since this toxin can cleave RNA in a ribosome-independent manner, this result was not anticipated and provided first evidence for the involvement of RF1 in the pathway of Kid toxicity. The sensitivity of the prfA mutants to RelE and Kid was restored to normal levels upon overproduction of the wild-type RF1 protein. We discuss these results and their utility for the design of novel antibacterial strategies in the light of the recently reported structure of ribosome-bound RF1.</description><identifier>ISSN: 0950-382X</identifier><identifier>EISSN: 1365-2958</identifier><identifier>DOI: 10.1111/j.1365-2958.2008.06510.x</identifier><identifier>PMID: 19019162</identifier><language>eng</language><publisher>Oxford, UK: Oxford, UK : Blackwell Publishing Ltd</publisher><subject>Bacterial Toxins - metabolism ; Bacteriology ; Biological and medical sciences ; Cellular biology ; Codon, Terminator ; E coli ; Escherichia coli ; Escherichia coli - genetics ; Escherichia coli - metabolism ; Escherichia coli Proteins - genetics ; Escherichia coli Proteins - metabolism ; Fundamental and applied biological sciences. Psychology ; Gene Expression Regulation, Bacterial ; Genetic Complementation Test ; Genetics ; Microbiology ; Miscellaneous ; Mutagenesis ; Mutation ; Peptide Chain Termination, Translational ; Peptide Termination Factors - genetics ; Peptide Termination Factors - metabolism ; Peptides ; Toxicity ; Toxins</subject><ispartof>Molecular microbiology, 2009-01, Vol.71 (1), p.66-78</ispartof><rights>2008 The Authors. Journal compilation © 2008 Blackwell Publishing Ltd</rights><rights>2009 INIST-CNRS</rights><rights>Copyright Blackwell Publishing Ltd. 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This original approach allowed the direct selection of mutants with altered translational termination efficiency at UAG codons. The isolated prfA mutants displayed a ~10-fold decrease in UAG termination efficiency with no significant changes in RF1 stability in vivo. All three mutations, G121S, G301S and R303H, were situated close to the nonsense codon recognition site in RF1:ribosome complexes. The prfA mutants displayed increased sensitivity to the RelE toxin encoded by the relBE system of E. coli, thus providing in vivo support for the functional interaction between RF1 and RelE. The prfA mutants also showed increased sensitivity to the Kid toxin. Since this toxin can cleave RNA in a ribosome-independent manner, this result was not anticipated and provided first evidence for the involvement of RF1 in the pathway of Kid toxicity. The sensitivity of the prfA mutants to RelE and Kid was restored to normal levels upon overproduction of the wild-type RF1 protein. We discuss these results and their utility for the design of novel antibacterial strategies in the light of the recently reported structure of ribosome-bound RF1.</description><subject>Bacterial Toxins - metabolism</subject><subject>Bacteriology</subject><subject>Biological and medical sciences</subject><subject>Cellular biology</subject><subject>Codon, Terminator</subject><subject>E coli</subject><subject>Escherichia coli</subject><subject>Escherichia coli - genetics</subject><subject>Escherichia coli - metabolism</subject><subject>Escherichia coli Proteins - genetics</subject><subject>Escherichia coli Proteins - metabolism</subject><subject>Fundamental and applied biological sciences. Psychology</subject><subject>Gene Expression Regulation, Bacterial</subject><subject>Genetic Complementation Test</subject><subject>Genetics</subject><subject>Microbiology</subject><subject>Miscellaneous</subject><subject>Mutagenesis</subject><subject>Mutation</subject><subject>Peptide Chain Termination, Translational</subject><subject>Peptide Termination Factors - genetics</subject><subject>Peptide Termination Factors - metabolism</subject><subject>Peptides</subject><subject>Toxicity</subject><subject>Toxins</subject><issn>0950-382X</issn><issn>1365-2958</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2009</creationdate><recordtype>article</recordtype><sourceid>24P</sourceid><sourceid>EIF</sourceid><recordid>eNqNkt1uEzEQhVcIRNPCK4CFBHcJ_ll71xdUQlUKFS1IhUrcWV6v3Thy7GI7afJIfUu8SQg_V-zNjvZ8c3ZGc6oKIDhB5Xk7nyDC6Bhz2k4whO0EMlq09aNqdBAeVyPIKRyTFn8_qo5TmkOICGTkaXWEOEQcMTyqHj6HlXZgmtRMR6tmVgIVnAXX5wgslln6nMC9zTPQaxW1TLoHOUqfnMw2eJB1XFi_q6XKdmXzBkjfA-t_4Un7ZPdKDiDPNFCbHHJYWwW0MVplEMz2e1csyhTSgUH1CXyy_dbtWrvps-qJkS7p5_v3SXVzPv129nF8-eXDxdn7y7GipIbjuqMdYaZBvMFcQtQzQikjdQ1NzfpSc2Zkh7jWuFNY9Qo1rDWm5cjIpuaYnFSnO9-7ZbfQvdK-LOzEXbQLGTciSCv-VrydiduwEpi1ELO6GLzZG8TwY6lTFgublHZOeh2WSWCIG4oxL-Crf8B5WEZflhOIM0qalsICtTtIxZBS1OYwCYJiCIOYi-HmYri5GMIgtmEQ69L64s9Nfjfur1-A13tAJiWdKYdVNh04XPLDSEsL927H3VunN_89gLi6uhiq0v9y129kEPI2ln_cfMVDGhFtKKoh-QngqNzZ</recordid><startdate>200901</startdate><enddate>200901</enddate><creator>Diago-Navarro, Elizabeth</creator><creator>Mora, Liliana</creator><creator>Buckingham, Richard H</creator><creator>Díaz-Orejas, Ramón</creator><creator>Lemonnier, Marc</creator><general>Oxford, UK : Blackwell Publishing Ltd</general><general>Blackwell Publishing Ltd</general><general>Blackwell</general><scope>FBQ</scope><scope>24P</scope><scope>IQODW</scope><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>7QL</scope><scope>7QP</scope><scope>7QR</scope><scope>7TK</scope><scope>7TM</scope><scope>7U9</scope><scope>8FD</scope><scope>C1K</scope><scope>FR3</scope><scope>H94</scope><scope>M7N</scope><scope>P64</scope><scope>RC3</scope><scope>7T7</scope><scope>7U7</scope><scope>5PM</scope></search><sort><creationdate>200901</creationdate><title>Novel Escherichia coli RF1 mutants with decreased translation termination activity and increased sensitivity to the cytotoxic effect of the bacterial toxins Kid and RelE</title><author>Diago-Navarro, Elizabeth ; Mora, Liliana ; Buckingham, Richard H ; Díaz-Orejas, Ramón ; Lemonnier, Marc</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c5340-4b5b36f719729a01d635563440f46d35596fab19ee2bc2cdc1768ff891fa74923</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2009</creationdate><topic>Bacterial Toxins - metabolism</topic><topic>Bacteriology</topic><topic>Biological and medical sciences</topic><topic>Cellular biology</topic><topic>Codon, Terminator</topic><topic>E coli</topic><topic>Escherichia coli</topic><topic>Escherichia coli - genetics</topic><topic>Escherichia coli - metabolism</topic><topic>Escherichia coli Proteins - genetics</topic><topic>Escherichia coli Proteins - metabolism</topic><topic>Fundamental and applied biological sciences. Psychology</topic><topic>Gene Expression Regulation, Bacterial</topic><topic>Genetic Complementation Test</topic><topic>Genetics</topic><topic>Microbiology</topic><topic>Miscellaneous</topic><topic>Mutagenesis</topic><topic>Mutation</topic><topic>Peptide Chain Termination, Translational</topic><topic>Peptide Termination Factors - genetics</topic><topic>Peptide Termination Factors - metabolism</topic><topic>Peptides</topic><topic>Toxicity</topic><topic>Toxins</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Diago-Navarro, Elizabeth</creatorcontrib><creatorcontrib>Mora, Liliana</creatorcontrib><creatorcontrib>Buckingham, Richard H</creatorcontrib><creatorcontrib>Díaz-Orejas, Ramón</creatorcontrib><creatorcontrib>Lemonnier, Marc</creatorcontrib><collection>AGRIS</collection><collection>Wiley Online Library Open Access</collection><collection>Pascal-Francis</collection><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>Bacteriology Abstracts (Microbiology B)</collection><collection>Calcium & Calcified Tissue Abstracts</collection><collection>Chemoreception Abstracts</collection><collection>Neurosciences Abstracts</collection><collection>Nucleic Acids Abstracts</collection><collection>Virology and AIDS Abstracts</collection><collection>Technology Research Database</collection><collection>Environmental Sciences and Pollution Management</collection><collection>Engineering Research Database</collection><collection>AIDS and Cancer Research Abstracts</collection><collection>Algology Mycology and Protozoology Abstracts (Microbiology C)</collection><collection>Biotechnology and BioEngineering Abstracts</collection><collection>Genetics Abstracts</collection><collection>Industrial and Applied Microbiology Abstracts (Microbiology A)</collection><collection>Toxicology Abstracts</collection><collection>PubMed Central (Full Participant titles)</collection><jtitle>Molecular microbiology</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Diago-Navarro, Elizabeth</au><au>Mora, Liliana</au><au>Buckingham, Richard H</au><au>Díaz-Orejas, Ramón</au><au>Lemonnier, Marc</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Novel Escherichia coli RF1 mutants with decreased translation termination activity and increased sensitivity to the cytotoxic effect of the bacterial toxins Kid and RelE</atitle><jtitle>Molecular microbiology</jtitle><addtitle>Mol Microbiol</addtitle><date>2009-01</date><risdate>2009</risdate><volume>71</volume><issue>1</issue><spage>66</spage><epage>78</epage><pages>66-78</pages><issn>0950-382X</issn><eissn>1365-2958</eissn><abstract>Novel mutations in prfA, the gene for the polypeptide release factor RF1 of Escherichia coli, were isolated using a positive genetic screen based on the parD (kis, kid) toxin-antitoxin system. This original approach allowed the direct selection of mutants with altered translational termination efficiency at UAG codons. The isolated prfA mutants displayed a ~10-fold decrease in UAG termination efficiency with no significant changes in RF1 stability in vivo. All three mutations, G121S, G301S and R303H, were situated close to the nonsense codon recognition site in RF1:ribosome complexes. The prfA mutants displayed increased sensitivity to the RelE toxin encoded by the relBE system of E. coli, thus providing in vivo support for the functional interaction between RF1 and RelE. The prfA mutants also showed increased sensitivity to the Kid toxin. Since this toxin can cleave RNA in a ribosome-independent manner, this result was not anticipated and provided first evidence for the involvement of RF1 in the pathway of Kid toxicity. The sensitivity of the prfA mutants to RelE and Kid was restored to normal levels upon overproduction of the wild-type RF1 protein. We discuss these results and their utility for the design of novel antibacterial strategies in the light of the recently reported structure of ribosome-bound RF1.</abstract><cop>Oxford, UK</cop><pub>Oxford, UK : Blackwell Publishing Ltd</pub><pmid>19019162</pmid><doi>10.1111/j.1365-2958.2008.06510.x</doi><tpages>13</tpages><oa>free_for_read</oa></addata></record> |
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| subjects | Bacterial Toxins - metabolism Bacteriology Biological and medical sciences Cellular biology Codon, Terminator E coli Escherichia coli Escherichia coli - genetics Escherichia coli - metabolism Escherichia coli Proteins - genetics Escherichia coli Proteins - metabolism Fundamental and applied biological sciences. Psychology Gene Expression Regulation, Bacterial Genetic Complementation Test Genetics Microbiology Miscellaneous Mutagenesis Mutation Peptide Chain Termination, Translational Peptide Termination Factors - genetics Peptide Termination Factors - metabolism Peptides Toxicity Toxins |
| title | Novel Escherichia coli RF1 mutants with decreased translation termination activity and increased sensitivity to the cytotoxic effect of the bacterial toxins Kid and RelE |
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