single-molecule assay for telomerase structure-function analysis
The activity of the telomerase ribonucleoprotein enzyme is essential for the maintenance of genome stability and normal cell development. Despite the biomedical importance of telomerase activity, detailed structural models for the enzyme remain to be established. Here we report a single-molecule ass...
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| Veröffentlicht in: | Nucleic acids research 2010-01, Vol.38 (3), p.e16-e16 |
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| container_title | Nucleic acids research |
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| creator | Wu, John Y Stone, Michael D Zhuang, Xiaowei |
| description | The activity of the telomerase ribonucleoprotein enzyme is essential for the maintenance of genome stability and normal cell development. Despite the biomedical importance of telomerase activity, detailed structural models for the enzyme remain to be established. Here we report a single-molecule assay for direct structural analysis of catalytically active telomerase enzymes. In this assay, oligonucleotide hybridization was used to probe the primer-extension activity of individual telomerase enzymes with single nucleotide sensitivity, allowing precise discrimination between inactive, active and processive enzyme binding events. FRET signals from enzyme molecules during the active and processive binding events were then used to determine the global organization of telomerase RNA within catalytically active holoenzymes. Using this assay, we have identified an active conformation of telomerase among a heterogeneous population of enzymes with distinct structures. |
| doi_str_mv | 10.1093/nar/gkp1033 |
| format | Article |
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Despite the biomedical importance of telomerase activity, detailed structural models for the enzyme remain to be established. Here we report a single-molecule assay for direct structural analysis of catalytically active telomerase enzymes. In this assay, oligonucleotide hybridization was used to probe the primer-extension activity of individual telomerase enzymes with single nucleotide sensitivity, allowing precise discrimination between inactive, active and processive enzyme binding events. FRET signals from enzyme molecules during the active and processive binding events were then used to determine the global organization of telomerase RNA within catalytically active holoenzymes. Using this assay, we have identified an active conformation of telomerase among a heterogeneous population of enzymes with distinct structures.</description><identifier>ISSN: 0305-1048</identifier><identifier>EISSN: 1362-4962</identifier><identifier>DOI: 10.1093/nar/gkp1033</identifier><identifier>PMID: 19920121</identifier><language>eng</language><publisher>England: Oxford University Press</publisher><subject>Biocatalysis ; Fluorescence Resonance Energy Transfer ; Methods Online ; Nucleic Acid Conformation ; Nucleic Acid Hybridization ; RNA - chemistry ; Telomerase - chemistry ; Telomerase - metabolism</subject><ispartof>Nucleic acids research, 2010-01, Vol.38 (3), p.e16-e16</ispartof><rights>The Author(s) 2009. 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Using this assay, we have identified an active conformation of telomerase among a heterogeneous population of enzymes with distinct structures.</description><subject>Biocatalysis</subject><subject>Fluorescence Resonance Energy Transfer</subject><subject>Methods Online</subject><subject>Nucleic Acid Conformation</subject><subject>Nucleic Acid Hybridization</subject><subject>RNA - chemistry</subject><subject>Telomerase - chemistry</subject><subject>Telomerase - metabolism</subject><issn>0305-1048</issn><issn>1362-4962</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2010</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNqFkb9v2zAQhYmiQe2knbIn2jIEqnkkJZNLkcDoL8BAhsQzcaJPjhJKdEkpgP_7yrCRplOmG96Hh3f4GDsH_hW4kbMO42zzvAUu5Qc2BVmKXJlSfGRTLnmRA1d6wk5TeuIcFBTqE5uAMYKDgCm7SU238ZS3wZMbPGWYEu6yOsSsJx9aipgoS30cXD9Eyuuhc30Tugw79LvUpM_spEaf6MvxnrHVj-8Pi1_58u7n78XtMneyLPvcEFYSEU0peelArVWh11gZsZaCHKAUGskACBRaaCewNkopCcpVpprXWp6xb4fe7VC1tHbU9RG93camxbizARv7f9I1j3YTXqzQMFclHwuujgUx_Bko9bZtkiPvsaMwJLuHdKGVfJ-UshAFn8NIXh9IF0NKkerXPcDtXo4d5dijnJG-ePvCP_ZoYwQuD0CNweImNsmu7sdIctBcmHHbXwFzllw</recordid><startdate>20100101</startdate><enddate>20100101</enddate><creator>Wu, John Y</creator><creator>Stone, Michael D</creator><creator>Zhuang, Xiaowei</creator><general>Oxford University Press</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>7X8</scope><scope>7TM</scope><scope>8FD</scope><scope>FR3</scope><scope>P64</scope><scope>RC3</scope><scope>5PM</scope></search><sort><creationdate>20100101</creationdate><title>single-molecule assay for telomerase structure-function analysis</title><author>Wu, John Y ; Stone, Michael D ; Zhuang, Xiaowei</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c366t-9eab3aaa96306c14d458dab92d32ec1a328ae9112a2828c2af9444314cb9b7f83</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2010</creationdate><topic>Biocatalysis</topic><topic>Fluorescence Resonance Energy Transfer</topic><topic>Methods Online</topic><topic>Nucleic Acid Conformation</topic><topic>Nucleic Acid Hybridization</topic><topic>RNA - chemistry</topic><topic>Telomerase - chemistry</topic><topic>Telomerase - metabolism</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Wu, John Y</creatorcontrib><creatorcontrib>Stone, Michael D</creatorcontrib><creatorcontrib>Zhuang, Xiaowei</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>MEDLINE - Academic</collection><collection>Nucleic Acids Abstracts</collection><collection>Technology Research Database</collection><collection>Engineering Research Database</collection><collection>Biotechnology and BioEngineering Abstracts</collection><collection>Genetics Abstracts</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>Wu, John Y</au><au>Stone, Michael D</au><au>Zhuang, Xiaowei</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>single-molecule assay for telomerase structure-function analysis</atitle><jtitle>Nucleic acids research</jtitle><addtitle>Nucleic Acids Res</addtitle><date>2010-01-01</date><risdate>2010</risdate><volume>38</volume><issue>3</issue><spage>e16</spage><epage>e16</epage><pages>e16-e16</pages><issn>0305-1048</issn><eissn>1362-4962</eissn><abstract>The activity of the telomerase ribonucleoprotein enzyme is essential for the maintenance of genome stability and normal cell development. 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| language | eng |
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| source | MEDLINE; DOAJ Directory of Open Access Journals; Oxford Journals Open Access Collection; PubMed Central; Free Full-Text Journals in Chemistry |
| subjects | Biocatalysis Fluorescence Resonance Energy Transfer Methods Online Nucleic Acid Conformation Nucleic Acid Hybridization RNA - chemistry Telomerase - chemistry Telomerase - metabolism |
| title | single-molecule assay for telomerase structure-function analysis |
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