Direct observation of a transient ternary complex during IκBα-mediated dissociation of NF-κB from DNA
We previously demonstrated that IκBα markedly increases the dissociation rate of DNA from NF-κB. The mechanism of this process remained a puzzle because no ternary complex was observed, and structures show that the DNA and IκBα binding sites on NF-κB are overlapping. The kinetics of interaction of I...
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Veröffentlicht in: | Proceedings of the National Academy of Sciences - PNAS 2014-01, Vol.111 (1), p.225-230 |
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description | We previously demonstrated that IκBα markedly increases the dissociation rate of DNA from NF-κB. The mechanism of this process remained a puzzle because no ternary complex was observed, and structures show that the DNA and IκBα binding sites on NF-κB are overlapping. The kinetics of interaction of IκBα with NF-κB and its complex with DNA were analyzed by using stopped-flow experiments in which fluorescence changes in pyrene-labeled DNA or the native tryptophan in IκBα were monitored. Rate constants governing the individual steps in the reaction were obtained from analysis of the measured rate vs. concentration profiles. The NF-κB association with DNA is extremely rapid with a rate constant of 1.5 × 10 ⁸ M ⁻¹⋅s ⁻¹. The NF-κB–DNA complex dissociates with a rate constant of 0.41 s ⁻¹, yielding a K D of 2.8 nM. When IκBα is added to the NF-κB–DNA complex, we observe the formation of a transient ternary complex in the first few milliseconds of the fluorescence trace, which rapidly rearranges to release DNA. The rate constant of this IκBα-mediated dissociation is nearly equal to the rate constant of association of IκBα with the NF-κB–DNA complex, showing that IκBα is optimized to repress transcription. The rate constants for the individual steps of a more folded mutant IκBα were also measured. This mutant associates with NF-κB more rapidly than wild-type IκBα, but it associates with the NF-κB–DNA complex more slowly and also is less efficient at mediating dissociation of the NF-κB–DNA complex. |
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The mechanism of this process remained a puzzle because no ternary complex was observed, and structures show that the DNA and IκBα binding sites on NF-κB are overlapping. The kinetics of interaction of IκBα with NF-κB and its complex with DNA were analyzed by using stopped-flow experiments in which fluorescence changes in pyrene-labeled DNA or the native tryptophan in IκBα were monitored. Rate constants governing the individual steps in the reaction were obtained from analysis of the measured rate vs. concentration profiles. The NF-κB association with DNA is extremely rapid with a rate constant of 1.5 × 10 ⁸ M ⁻¹⋅s ⁻¹. The NF-κB–DNA complex dissociates with a rate constant of 0.41 s ⁻¹, yielding a K D of 2.8 nM. When IκBα is added to the NF-κB–DNA complex, we observe the formation of a transient ternary complex in the first few milliseconds of the fluorescence trace, which rapidly rearranges to release DNA. The rate constant of this IκBα-mediated dissociation is nearly equal to the rate constant of association of IκBα with the NF-κB–DNA complex, showing that IκBα is optimized to repress transcription. The rate constants for the individual steps of a more folded mutant IκBα were also measured. This mutant associates with NF-κB more rapidly than wild-type IκBα, but it associates with the NF-κB–DNA complex more slowly and also is less efficient at mediating dissociation of the NF-κB–DNA complex.</description><identifier>ISSN: 0027-8424</identifier><identifier>EISSN: 1091-6490</identifier><identifier>DOI: 10.1073/pnas.1318115111</identifier><identifier>PMID: 24367071</identifier><language>eng</language><publisher>United States: National Academy of Sciences</publisher><subject>Association reactions ; Binding sites ; Biological Sciences ; Cell Nucleus - metabolism ; DNA ; DNA - chemistry ; Dose-Response Relationship, Drug ; Fluorescence ; Fluorescent Dyes - chemistry ; Gene Expression Regulation ; Genes ; Health care industry ; Humans ; I-kappa B Proteins - genetics ; I-kappa B Proteins - metabolism ; Kinetics ; Microscopy, Fluorescence ; Molecules ; Mutation ; NF-kappa B p50 Subunit - genetics ; NF-kappa B p50 Subunit - metabolism ; NF-KappaB Inhibitor alpha ; Pests ; Protein Binding ; Protein Conformation ; Proteins ; Pyrenes - chemistry ; Signal Transduction ; Time Factors ; Transcription, Genetic ; Transcriptional Activation ; Tryptophan - chemistry</subject><ispartof>Proceedings of the National Academy of Sciences - PNAS, 2014-01, Vol.111 (1), p.225-230</ispartof><rights>copyright © 1993—2008 National Academy of Sciences of the United States of America</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c494t-1b5a02165c03aad4c24c9ab5d91cb3dc184dce824781aafce5e68b813b8725ce3</citedby><cites>FETCH-LOGICAL-c494t-1b5a02165c03aad4c24c9ab5d91cb3dc184dce824781aafce5e68b813b8725ce3</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Uhttp://www.pnas.org/content/111/1.cover.gif</thumbnail><linktopdf>$$Uhttps://www.jstor.org/stable/pdf/23770526$$EPDF$$P50$$Gjstor$$H</linktopdf><linktohtml>$$Uhttps://www.jstor.org/stable/23770526$$EHTML$$P50$$Gjstor$$H</linktohtml><link.rule.ids>230,314,727,780,784,803,885,27924,27925,53791,53793,58017,58250</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/24367071$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Alverdi, Vera</creatorcontrib><creatorcontrib>Hetrick, Byron</creatorcontrib><creatorcontrib>Joseph, Simpson</creatorcontrib><creatorcontrib>Komives, Elizabeth A.</creatorcontrib><title>Direct observation of a transient ternary complex during IκBα-mediated dissociation of NF-κB from DNA</title><title>Proceedings of the National Academy of Sciences - PNAS</title><addtitle>Proc Natl Acad Sci U S A</addtitle><description>We previously demonstrated that IκBα markedly increases the dissociation rate of DNA from NF-κB. The mechanism of this process remained a puzzle because no ternary complex was observed, and structures show that the DNA and IκBα binding sites on NF-κB are overlapping. The kinetics of interaction of IκBα with NF-κB and its complex with DNA were analyzed by using stopped-flow experiments in which fluorescence changes in pyrene-labeled DNA or the native tryptophan in IκBα were monitored. Rate constants governing the individual steps in the reaction were obtained from analysis of the measured rate vs. concentration profiles. The NF-κB association with DNA is extremely rapid with a rate constant of 1.5 × 10 ⁸ M ⁻¹⋅s ⁻¹. The NF-κB–DNA complex dissociates with a rate constant of 0.41 s ⁻¹, yielding a K D of 2.8 nM. When IκBα is added to the NF-κB–DNA complex, we observe the formation of a transient ternary complex in the first few milliseconds of the fluorescence trace, which rapidly rearranges to release DNA. The rate constant of this IκBα-mediated dissociation is nearly equal to the rate constant of association of IκBα with the NF-κB–DNA complex, showing that IκBα is optimized to repress transcription. The rate constants for the individual steps of a more folded mutant IκBα were also measured. This mutant associates with NF-κB more rapidly than wild-type IκBα, but it associates with the NF-κB–DNA complex more slowly and also is less efficient at mediating dissociation of the NF-κB–DNA complex.</description><subject>Association reactions</subject><subject>Binding sites</subject><subject>Biological Sciences</subject><subject>Cell Nucleus - metabolism</subject><subject>DNA</subject><subject>DNA - chemistry</subject><subject>Dose-Response Relationship, Drug</subject><subject>Fluorescence</subject><subject>Fluorescent Dyes - chemistry</subject><subject>Gene Expression Regulation</subject><subject>Genes</subject><subject>Health care industry</subject><subject>Humans</subject><subject>I-kappa B Proteins - genetics</subject><subject>I-kappa B Proteins - metabolism</subject><subject>Kinetics</subject><subject>Microscopy, Fluorescence</subject><subject>Molecules</subject><subject>Mutation</subject><subject>NF-kappa B p50 Subunit - genetics</subject><subject>NF-kappa B p50 Subunit - metabolism</subject><subject>NF-KappaB Inhibitor alpha</subject><subject>Pests</subject><subject>Protein Binding</subject><subject>Protein Conformation</subject><subject>Proteins</subject><subject>Pyrenes - chemistry</subject><subject>Signal Transduction</subject><subject>Time Factors</subject><subject>Transcription, Genetic</subject><subject>Transcriptional Activation</subject><subject>Tryptophan - chemistry</subject><issn>0027-8424</issn><issn>1091-6490</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2014</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNpVkc1u1DAUhS0EokNhzQrwkk3ae_0TOxuk_lOpKgvo2nIcZ-oqiQc7U8FjseUh-kzNaKYz7eouznfOvVeHkI8IBwiKHy4Gmw-Qo0aUiPiKzBAqLEpRwWsyA2Cq0IKJPfIu5zsAqKSGt2SPCV4qUDgjt6cheTfSWGef7u0Y4kBjSy0dkx1y8MNIR58Gm_5SF_tF5__QZpnCMKeXD_-PH_4VvW-CHX1Dm5BzdGEbcX1eTARtU-zp6fXRe_KmtV32HzZzn9ycn_06-V5c_bi4PDm6KpyoxFhgLS0wLKUDbm0jHBOusrVsKnQ1bxxq0TivmVAarW2dl77UtUZea8Wk83yffFvnLpb1dJubPki2M4sU-ukJE20wL5Uh3Jp5vDdcV6AUmwK-bgJS_L30eTR9yM53nR18XGaDEiTnrOJ8Qg_XqEsx5-Tb7RoEs-rHrPoxu34mx-fn1235p0Im4NMGWDm3cYgGDWNyp9_lMaadnysFkpWT_mWttzYaO08hm5ufDLAEQME1AH8EmXisjA</recordid><startdate>20140107</startdate><enddate>20140107</enddate><creator>Alverdi, Vera</creator><creator>Hetrick, Byron</creator><creator>Joseph, Simpson</creator><creator>Komives, Elizabeth A.</creator><general>National Academy of Sciences</general><general>National Acad Sciences</general><scope>FBQ</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>7TM</scope><scope>5PM</scope></search><sort><creationdate>20140107</creationdate><title>Direct observation of a transient ternary complex during IκBα-mediated dissociation of NF-κB from DNA</title><author>Alverdi, Vera ; Hetrick, Byron ; Joseph, Simpson ; Komives, Elizabeth A.</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c494t-1b5a02165c03aad4c24c9ab5d91cb3dc184dce824781aafce5e68b813b8725ce3</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2014</creationdate><topic>Association reactions</topic><topic>Binding sites</topic><topic>Biological Sciences</topic><topic>Cell Nucleus - metabolism</topic><topic>DNA</topic><topic>DNA - chemistry</topic><topic>Dose-Response Relationship, Drug</topic><topic>Fluorescence</topic><topic>Fluorescent Dyes - chemistry</topic><topic>Gene Expression Regulation</topic><topic>Genes</topic><topic>Health care industry</topic><topic>Humans</topic><topic>I-kappa B Proteins - genetics</topic><topic>I-kappa B Proteins - metabolism</topic><topic>Kinetics</topic><topic>Microscopy, Fluorescence</topic><topic>Molecules</topic><topic>Mutation</topic><topic>NF-kappa B p50 Subunit - genetics</topic><topic>NF-kappa B p50 Subunit - metabolism</topic><topic>NF-KappaB Inhibitor alpha</topic><topic>Pests</topic><topic>Protein Binding</topic><topic>Protein Conformation</topic><topic>Proteins</topic><topic>Pyrenes - chemistry</topic><topic>Signal Transduction</topic><topic>Time Factors</topic><topic>Transcription, Genetic</topic><topic>Transcriptional Activation</topic><topic>Tryptophan - chemistry</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Alverdi, Vera</creatorcontrib><creatorcontrib>Hetrick, Byron</creatorcontrib><creatorcontrib>Joseph, Simpson</creatorcontrib><creatorcontrib>Komives, Elizabeth A.</creatorcontrib><collection>AGRIS</collection><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>Nucleic Acids Abstracts</collection><collection>PubMed Central (Full Participant titles)</collection><jtitle>Proceedings of the National Academy of Sciences - PNAS</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Alverdi, Vera</au><au>Hetrick, Byron</au><au>Joseph, Simpson</au><au>Komives, Elizabeth A.</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Direct observation of a transient ternary complex during IκBα-mediated dissociation of NF-κB from DNA</atitle><jtitle>Proceedings of the National Academy of Sciences - PNAS</jtitle><addtitle>Proc Natl Acad Sci U S A</addtitle><date>2014-01-07</date><risdate>2014</risdate><volume>111</volume><issue>1</issue><spage>225</spage><epage>230</epage><pages>225-230</pages><issn>0027-8424</issn><eissn>1091-6490</eissn><abstract>We previously demonstrated that IκBα markedly increases the dissociation rate of DNA from NF-κB. The mechanism of this process remained a puzzle because no ternary complex was observed, and structures show that the DNA and IκBα binding sites on NF-κB are overlapping. The kinetics of interaction of IκBα with NF-κB and its complex with DNA were analyzed by using stopped-flow experiments in which fluorescence changes in pyrene-labeled DNA or the native tryptophan in IκBα were monitored. Rate constants governing the individual steps in the reaction were obtained from analysis of the measured rate vs. concentration profiles. The NF-κB association with DNA is extremely rapid with a rate constant of 1.5 × 10 ⁸ M ⁻¹⋅s ⁻¹. The NF-κB–DNA complex dissociates with a rate constant of 0.41 s ⁻¹, yielding a K D of 2.8 nM. When IκBα is added to the NF-κB–DNA complex, we observe the formation of a transient ternary complex in the first few milliseconds of the fluorescence trace, which rapidly rearranges to release DNA. The rate constant of this IκBα-mediated dissociation is nearly equal to the rate constant of association of IκBα with the NF-κB–DNA complex, showing that IκBα is optimized to repress transcription. The rate constants for the individual steps of a more folded mutant IκBα were also measured. This mutant associates with NF-κB more rapidly than wild-type IκBα, but it associates with the NF-κB–DNA complex more slowly and also is less efficient at mediating dissociation of the NF-κB–DNA complex.</abstract><cop>United States</cop><pub>National Academy of Sciences</pub><pmid>24367071</pmid><doi>10.1073/pnas.1318115111</doi><tpages>6</tpages><oa>free_for_read</oa></addata></record> |
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subjects | Association reactions Binding sites Biological Sciences Cell Nucleus - metabolism DNA DNA - chemistry Dose-Response Relationship, Drug Fluorescence Fluorescent Dyes - chemistry Gene Expression Regulation Genes Health care industry Humans I-kappa B Proteins - genetics I-kappa B Proteins - metabolism Kinetics Microscopy, Fluorescence Molecules Mutation NF-kappa B p50 Subunit - genetics NF-kappa B p50 Subunit - metabolism NF-KappaB Inhibitor alpha Pests Protein Binding Protein Conformation Proteins Pyrenes - chemistry Signal Transduction Time Factors Transcription, Genetic Transcriptional Activation Tryptophan - chemistry |
title | Direct observation of a transient ternary complex during IκBα-mediated dissociation of NF-κB from DNA |
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