RNA polymerase clamp conformational dynamics: long-lived states and modulation by crowding, cations, and nonspecific DNA binding
Abstract The RNA polymerase (RNAP) clamp, a mobile structural element conserved in RNAP from all domains of life, has been proposed to play critical roles at different stages of transcription. In previous work, we demonstrated using single-molecule Förster resonance energy transfer (smFRET) that RNA...
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Veröffentlicht in: | Nucleic acids research 2021-03, Vol.49 (5), p.2790-2802 |
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creator | Mazumder, Abhishek Wang, Anna Uhm, Heesoo Ebright, Richard H Kapanidis, Achillefs N |
description | Abstract
The RNA polymerase (RNAP) clamp, a mobile structural element conserved in RNAP from all domains of life, has been proposed to play critical roles at different stages of transcription. In previous work, we demonstrated using single-molecule Förster resonance energy transfer (smFRET) that RNAP clamp interconvert between three short-lived conformational states (lifetimes ∼ 0.3–0.6 s), that the clamp can be locked into any one of these states by small molecules, and that the clamp stays closed during initial transcription and elongation. Here, we extend these studies to obtain a comprehensive understanding of clamp dynamics under conditions RNAP may encounter in living cells. We find that the RNAP clamp can populate long-lived conformational states (lifetimes > 1.0 s) and can switch between these long-lived states and the previously observed short-lived states. In addition, we find that clamp motions are increased in the presence of molecular crowding, are unchanged in the presence of elevated monovalent-cation concentrations, and are reduced in the presence of elevated divalent-cation concentrations. Finally, we find that RNAP bound to non-specific DNA predominantly exhibits a closed clamp conformation. Our results raise the possibility of additional regulatory checkpoints that could affect clamp dynamics and consequently could affect transcription and transcriptional regulation. |
doi_str_mv | 10.1093/nar/gkab074 |
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The RNA polymerase (RNAP) clamp, a mobile structural element conserved in RNAP from all domains of life, has been proposed to play critical roles at different stages of transcription. In previous work, we demonstrated using single-molecule Förster resonance energy transfer (smFRET) that RNAP clamp interconvert between three short-lived conformational states (lifetimes ∼ 0.3–0.6 s), that the clamp can be locked into any one of these states by small molecules, and that the clamp stays closed during initial transcription and elongation. Here, we extend these studies to obtain a comprehensive understanding of clamp dynamics under conditions RNAP may encounter in living cells. We find that the RNAP clamp can populate long-lived conformational states (lifetimes > 1.0 s) and can switch between these long-lived states and the previously observed short-lived states. In addition, we find that clamp motions are increased in the presence of molecular crowding, are unchanged in the presence of elevated monovalent-cation concentrations, and are reduced in the presence of elevated divalent-cation concentrations. Finally, we find that RNAP bound to non-specific DNA predominantly exhibits a closed clamp conformation. Our results raise the possibility of additional regulatory checkpoints that could affect clamp dynamics and consequently could affect transcription and transcriptional regulation.</description><identifier>ISSN: 0305-1048</identifier><identifier>EISSN: 1362-4962</identifier><identifier>DOI: 10.1093/nar/gkab074</identifier><identifier>PMID: 33589919</identifier><language>eng</language><publisher>England: Oxford University Press</publisher><subject>Cations, Divalent ; Cations, Monovalent ; DNA - metabolism ; DNA-Directed RNA Polymerases - chemistry ; DNA-Directed RNA Polymerases - metabolism ; Escherichia coli - enzymology ; Fluorescence Resonance Energy Transfer ; Nucleic Acid Enzymes ; Polyethylene Glycols - chemistry ; Protein Conformation</subject><ispartof>Nucleic acids research, 2021-03, Vol.49 (5), p.2790-2802</ispartof><rights>The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research. 2021</rights><rights>The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research.</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c412t-2e66072b29c743b78c7706d3c41279adeedd85f9f7d3ad56a947876508275ec33</citedby><cites>FETCH-LOGICAL-c412t-2e66072b29c743b78c7706d3c41279adeedd85f9f7d3ad56a947876508275ec33</cites><orcidid>0000-0002-9339-6256</orcidid></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC7969002/pdf/$$EPDF$$P50$$Gpubmedcentral$$Hfree_for_read</linktopdf><linktohtml>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC7969002/$$EHTML$$P50$$Gpubmedcentral$$Hfree_for_read</linktohtml><link.rule.ids>230,314,723,776,780,860,881,1598,27901,27902,53766,53768</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/33589919$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Mazumder, Abhishek</creatorcontrib><creatorcontrib>Wang, Anna</creatorcontrib><creatorcontrib>Uhm, Heesoo</creatorcontrib><creatorcontrib>Ebright, Richard H</creatorcontrib><creatorcontrib>Kapanidis, Achillefs N</creatorcontrib><title>RNA polymerase clamp conformational dynamics: long-lived states and modulation by crowding, cations, and nonspecific DNA binding</title><title>Nucleic acids research</title><addtitle>Nucleic Acids Res</addtitle><description>Abstract
The RNA polymerase (RNAP) clamp, a mobile structural element conserved in RNAP from all domains of life, has been proposed to play critical roles at different stages of transcription. In previous work, we demonstrated using single-molecule Förster resonance energy transfer (smFRET) that RNAP clamp interconvert between three short-lived conformational states (lifetimes ∼ 0.3–0.6 s), that the clamp can be locked into any one of these states by small molecules, and that the clamp stays closed during initial transcription and elongation. Here, we extend these studies to obtain a comprehensive understanding of clamp dynamics under conditions RNAP may encounter in living cells. We find that the RNAP clamp can populate long-lived conformational states (lifetimes > 1.0 s) and can switch between these long-lived states and the previously observed short-lived states. In addition, we find that clamp motions are increased in the presence of molecular crowding, are unchanged in the presence of elevated monovalent-cation concentrations, and are reduced in the presence of elevated divalent-cation concentrations. Finally, we find that RNAP bound to non-specific DNA predominantly exhibits a closed clamp conformation. Our results raise the possibility of additional regulatory checkpoints that could affect clamp dynamics and consequently could affect transcription and transcriptional regulation.</description><subject>Cations, Divalent</subject><subject>Cations, Monovalent</subject><subject>DNA - metabolism</subject><subject>DNA-Directed RNA Polymerases - chemistry</subject><subject>DNA-Directed RNA Polymerases - metabolism</subject><subject>Escherichia coli - enzymology</subject><subject>Fluorescence Resonance Energy Transfer</subject><subject>Nucleic Acid Enzymes</subject><subject>Polyethylene Glycols - chemistry</subject><subject>Protein Conformation</subject><issn>0305-1048</issn><issn>1362-4962</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2021</creationdate><recordtype>article</recordtype><sourceid>TOX</sourceid><sourceid>EIF</sourceid><recordid>eNp9kUtv1TAQhS1ERS-FFXvkFUKioX4ljlkgVQVapAokBGtrYjsXg2OndlJ0d_3p5D6oYMNqRnM-nRnNQegZJa8pUfwsQj5b_4SOSPEArShvWCVUwx6iFeGkrigR7TF6XMoPQqigtXiEjjmvW6WoWqG7L5_O8ZjCZnAZisMmwDBik2Kf8gCTTxECtpsIgzflDQ4prqvgb53FZYLJFQzR4iHZOexg3G2wyemX9XF9is1uVk53UFy60Rnfe4PfLUs7H7fUE3TUQyju6aGeoG8f3n-9uKquP19-vDi_roygbKqYaxoiWceUkYJ3sjVSksbyrSoVWOesbete9dJysHUDSshWNjVpmayd4fwEvd37jnM3OGtcnDIEPWY_QN7oBF7_q0T_Xa_TrZaqUYSwxeDlwSCnm9mVSQ--GBcCRJfmoplQhLJa0HZBX-3R5RWlZNffr6FEbzPTS2b6kNlCP__7snv2T0gL8GIPpHn8r9NvxPmjLg</recordid><startdate>20210318</startdate><enddate>20210318</enddate><creator>Mazumder, Abhishek</creator><creator>Wang, Anna</creator><creator>Uhm, Heesoo</creator><creator>Ebright, Richard H</creator><creator>Kapanidis, Achillefs N</creator><general>Oxford University Press</general><scope>TOX</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>7X8</scope><scope>5PM</scope><orcidid>https://orcid.org/0000-0002-9339-6256</orcidid></search><sort><creationdate>20210318</creationdate><title>RNA polymerase clamp conformational dynamics: long-lived states and modulation by crowding, cations, and nonspecific DNA binding</title><author>Mazumder, Abhishek ; Wang, Anna ; Uhm, Heesoo ; Ebright, Richard H ; Kapanidis, Achillefs N</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c412t-2e66072b29c743b78c7706d3c41279adeedd85f9f7d3ad56a947876508275ec33</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2021</creationdate><topic>Cations, Divalent</topic><topic>Cations, Monovalent</topic><topic>DNA - metabolism</topic><topic>DNA-Directed RNA Polymerases - chemistry</topic><topic>DNA-Directed RNA Polymerases - metabolism</topic><topic>Escherichia coli - enzymology</topic><topic>Fluorescence Resonance Energy Transfer</topic><topic>Nucleic Acid Enzymes</topic><topic>Polyethylene Glycols - chemistry</topic><topic>Protein Conformation</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Mazumder, Abhishek</creatorcontrib><creatorcontrib>Wang, Anna</creatorcontrib><creatorcontrib>Uhm, Heesoo</creatorcontrib><creatorcontrib>Ebright, Richard H</creatorcontrib><creatorcontrib>Kapanidis, Achillefs N</creatorcontrib><collection>Oxford Journals Open Access Collection</collection><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>MEDLINE - Academic</collection><collection>PubMed Central (Full Participant titles)</collection><jtitle>Nucleic acids research</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Mazumder, Abhishek</au><au>Wang, Anna</au><au>Uhm, Heesoo</au><au>Ebright, Richard H</au><au>Kapanidis, Achillefs N</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>RNA polymerase clamp conformational dynamics: long-lived states and modulation by crowding, cations, and nonspecific DNA binding</atitle><jtitle>Nucleic acids research</jtitle><addtitle>Nucleic Acids Res</addtitle><date>2021-03-18</date><risdate>2021</risdate><volume>49</volume><issue>5</issue><spage>2790</spage><epage>2802</epage><pages>2790-2802</pages><issn>0305-1048</issn><eissn>1362-4962</eissn><abstract>Abstract
The RNA polymerase (RNAP) clamp, a mobile structural element conserved in RNAP from all domains of life, has been proposed to play critical roles at different stages of transcription. In previous work, we demonstrated using single-molecule Förster resonance energy transfer (smFRET) that RNAP clamp interconvert between three short-lived conformational states (lifetimes ∼ 0.3–0.6 s), that the clamp can be locked into any one of these states by small molecules, and that the clamp stays closed during initial transcription and elongation. Here, we extend these studies to obtain a comprehensive understanding of clamp dynamics under conditions RNAP may encounter in living cells. We find that the RNAP clamp can populate long-lived conformational states (lifetimes > 1.0 s) and can switch between these long-lived states and the previously observed short-lived states. In addition, we find that clamp motions are increased in the presence of molecular crowding, are unchanged in the presence of elevated monovalent-cation concentrations, and are reduced in the presence of elevated divalent-cation concentrations. Finally, we find that RNAP bound to non-specific DNA predominantly exhibits a closed clamp conformation. Our results raise the possibility of additional regulatory checkpoints that could affect clamp dynamics and consequently could affect transcription and transcriptional regulation.</abstract><cop>England</cop><pub>Oxford University Press</pub><pmid>33589919</pmid><doi>10.1093/nar/gkab074</doi><tpages>13</tpages><orcidid>https://orcid.org/0000-0002-9339-6256</orcidid><oa>free_for_read</oa></addata></record> |
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subjects | Cations, Divalent Cations, Monovalent DNA - metabolism DNA-Directed RNA Polymerases - chemistry DNA-Directed RNA Polymerases - metabolism Escherichia coli - enzymology Fluorescence Resonance Energy Transfer Nucleic Acid Enzymes Polyethylene Glycols - chemistry Protein Conformation |
title | RNA polymerase clamp conformational dynamics: long-lived states and modulation by crowding, cations, and nonspecific DNA binding |
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