Molecular Mechanism of Transcription Inhibition by Peptide Antibiotic Microcin J25
21 amino acid peptide Microcin J25 (MccJ25) inhibits transcription by bacterial RNA polymerase (RNAP). MccJ25-resistance mutations cluster in the RNAP secondary channel through which incoming NTP substrates are thought to reach the catalytic center and the 3′ end of the nascent RNA is likely to thre...
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| Veröffentlicht in: | Molecular cell 2004-06, Vol.14 (6), p.753-762 |
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| container_title | Molecular cell |
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| creator | Adelman, Karen Yuzenkova, Julia La Porta, Arthur Zenkin, Nikolay Lee, Jookyung Lis, John T Borukhov, Sergei Wang, Michelle D Severinov, Konstantin |
| description | 21 amino acid peptide Microcin J25 (MccJ25) inhibits transcription by bacterial RNA polymerase (RNAP). MccJ25-resistance mutations cluster in the RNAP secondary channel through which incoming NTP substrates are thought to reach the catalytic center and the 3′ end of the nascent RNA is likely to thread in backtracked transcription complexes. The secondary channel also accepts transcript cleavage factors GreA and GreB. Here, we demonstrate that MccJ25 inhibits GreA/GreB-dependent transcript cleavage, impedes formation of backtracked complexes, and can be crosslinked to the 3′-end of the nascent RNA in elongation complexes. These results place the MccJ25 binding site within the secondary channel. Moreover, single-molecule assays reveal that MccJ25 binding to a transcribing RNAP temporarily stops transcript elongation but has no effect on the elongation velocity between pauses. Kinetic analysis of single-molecule data allows us to put forward a model of transcription inhibition by MccJ25 that envisions the complete occlusion of the secondary channel by bound inhibitor. |
| doi_str_mv | 10.1016/j.molcel.2004.05.017 |
| format | Article |
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MccJ25-resistance mutations cluster in the RNAP secondary channel through which incoming NTP substrates are thought to reach the catalytic center and the 3′ end of the nascent RNA is likely to thread in backtracked transcription complexes. The secondary channel also accepts transcript cleavage factors GreA and GreB. Here, we demonstrate that MccJ25 inhibits GreA/GreB-dependent transcript cleavage, impedes formation of backtracked complexes, and can be crosslinked to the 3′-end of the nascent RNA in elongation complexes. These results place the MccJ25 binding site within the secondary channel. Moreover, single-molecule assays reveal that MccJ25 binding to a transcribing RNAP temporarily stops transcript elongation but has no effect on the elongation velocity between pauses. Kinetic analysis of single-molecule data allows us to put forward a model of transcription inhibition by MccJ25 that envisions the complete occlusion of the secondary channel by bound inhibitor.</description><identifier>ISSN: 1097-2765</identifier><identifier>EISSN: 1097-4164</identifier><identifier>DOI: 10.1016/j.molcel.2004.05.017</identifier><identifier>PMID: 15200953</identifier><language>eng</language><publisher>United States: Elsevier Inc</publisher><subject>Anti-Bacterial Agents - pharmacology ; Bacteriocins - analysis ; Bacteriocins - metabolism ; Bacteriocins - pharmacology ; DNA-Directed RNA Polymerases - antagonists & inhibitors ; DNA-Directed RNA Polymerases - chemistry ; DNA-Directed RNA Polymerases - metabolism ; Kinetics ; Models, Molecular ; Peptides - pharmacology ; Transcription, Genetic - drug effects ; Transcriptional Elongation Factors - physiology</subject><ispartof>Molecular cell, 2004-06, Vol.14 (6), p.753-762</ispartof><rights>2004 Cell Press</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c501t-90b10259637870ac4f6f561b4ee6fb77c8dd7b95a762de1eabf8474086f3d4573</citedby><cites>FETCH-LOGICAL-c501t-90b10259637870ac4f6f561b4ee6fb77c8dd7b95a762de1eabf8474086f3d4573</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktohtml>$$Uhttps://dx.doi.org/10.1016/j.molcel.2004.05.017$$EHTML$$P50$$Gelsevier$$Hfree_for_read</linktohtml><link.rule.ids>314,780,784,3550,27924,27925,45995</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/15200953$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Adelman, Karen</creatorcontrib><creatorcontrib>Yuzenkova, Julia</creatorcontrib><creatorcontrib>La Porta, Arthur</creatorcontrib><creatorcontrib>Zenkin, Nikolay</creatorcontrib><creatorcontrib>Lee, Jookyung</creatorcontrib><creatorcontrib>Lis, John T</creatorcontrib><creatorcontrib>Borukhov, Sergei</creatorcontrib><creatorcontrib>Wang, Michelle D</creatorcontrib><creatorcontrib>Severinov, Konstantin</creatorcontrib><title>Molecular Mechanism of Transcription Inhibition by Peptide Antibiotic Microcin J25</title><title>Molecular cell</title><addtitle>Mol Cell</addtitle><description>21 amino acid peptide Microcin J25 (MccJ25) inhibits transcription by bacterial RNA polymerase (RNAP). MccJ25-resistance mutations cluster in the RNAP secondary channel through which incoming NTP substrates are thought to reach the catalytic center and the 3′ end of the nascent RNA is likely to thread in backtracked transcription complexes. The secondary channel also accepts transcript cleavage factors GreA and GreB. Here, we demonstrate that MccJ25 inhibits GreA/GreB-dependent transcript cleavage, impedes formation of backtracked complexes, and can be crosslinked to the 3′-end of the nascent RNA in elongation complexes. These results place the MccJ25 binding site within the secondary channel. Moreover, single-molecule assays reveal that MccJ25 binding to a transcribing RNAP temporarily stops transcript elongation but has no effect on the elongation velocity between pauses. Kinetic analysis of single-molecule data allows us to put forward a model of transcription inhibition by MccJ25 that envisions the complete occlusion of the secondary channel by bound inhibitor.</description><subject>Anti-Bacterial Agents - pharmacology</subject><subject>Bacteriocins - analysis</subject><subject>Bacteriocins - metabolism</subject><subject>Bacteriocins - pharmacology</subject><subject>DNA-Directed RNA Polymerases - antagonists & inhibitors</subject><subject>DNA-Directed RNA Polymerases - chemistry</subject><subject>DNA-Directed RNA Polymerases - metabolism</subject><subject>Kinetics</subject><subject>Models, Molecular</subject><subject>Peptides - pharmacology</subject><subject>Transcription, Genetic - drug effects</subject><subject>Transcriptional Elongation Factors - physiology</subject><issn>1097-2765</issn><issn>1097-4164</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2004</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNqFkF1LwzAUhoMobk7_gUivvFtN2ny0N8IYfkw2FJnXIU1PWUbbzKQT9u_NbME7vTqHw_OekzwIXRMcE0z43TZubK2hjhOMaYxZjIk4QWOCczGlhNPToU8EZyN04f0WY0JZlp-jEWEhlLN0jN5Xtga9r5WLVqA3qjW-iWwVrZ1qvXZm1xnbRot2Ywrz0xaH6A3CtIRo1nZhajujo5XRzmrTRi8Ju0Rnlao9XA11gj4eH9bz5-ny9Wkxny2nmmHSTXNcEJywnKciE1hpWvGKcVJQAF4VQuisLEWRMyV4UgIBVVQZFRRnvEpLykQ6Qbf93p2zn3vwnWyMD0Jq1YLde8k5TzNCkn9BIvIsCy8JIO3B8BnvHVRy50yj3EESLI_S5Vb20uVRusRMBukhdjPs3xcNlL-hwXIA7nsAgo4vA056baDVUBoHupOlNX9f-AbS_JRE</recordid><startdate>20040618</startdate><enddate>20040618</enddate><creator>Adelman, Karen</creator><creator>Yuzenkova, Julia</creator><creator>La Porta, Arthur</creator><creator>Zenkin, Nikolay</creator><creator>Lee, Jookyung</creator><creator>Lis, John T</creator><creator>Borukhov, Sergei</creator><creator>Wang, Michelle D</creator><creator>Severinov, Konstantin</creator><general>Elsevier Inc</general><scope>6I.</scope><scope>AAFTH</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>7TM</scope><scope>C1K</scope><scope>7X8</scope></search><sort><creationdate>20040618</creationdate><title>Molecular Mechanism of Transcription Inhibition by Peptide Antibiotic Microcin J25</title><author>Adelman, Karen ; 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MccJ25-resistance mutations cluster in the RNAP secondary channel through which incoming NTP substrates are thought to reach the catalytic center and the 3′ end of the nascent RNA is likely to thread in backtracked transcription complexes. The secondary channel also accepts transcript cleavage factors GreA and GreB. Here, we demonstrate that MccJ25 inhibits GreA/GreB-dependent transcript cleavage, impedes formation of backtracked complexes, and can be crosslinked to the 3′-end of the nascent RNA in elongation complexes. These results place the MccJ25 binding site within the secondary channel. Moreover, single-molecule assays reveal that MccJ25 binding to a transcribing RNAP temporarily stops transcript elongation but has no effect on the elongation velocity between pauses. 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| subjects | Anti-Bacterial Agents - pharmacology Bacteriocins - analysis Bacteriocins - metabolism Bacteriocins - pharmacology DNA-Directed RNA Polymerases - antagonists & inhibitors DNA-Directed RNA Polymerases - chemistry DNA-Directed RNA Polymerases - metabolism Kinetics Models, Molecular Peptides - pharmacology Transcription, Genetic - drug effects Transcriptional Elongation Factors - physiology |
| title | Molecular Mechanism of Transcription Inhibition by Peptide Antibiotic Microcin J25 |
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