Single-Molecule Real-Time 3D Imaging of the Transcription Cycle by Modulation Interferometry
Many essential cellular processes, such as gene control, employ elaborate mechanisms involving the coordination of large, multi-component molecular assemblies. Few structural biology tools presently have the combined spatial-temporal resolution and molecular specificity required to capture the movem...
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| Veröffentlicht in: | Cell 2016-12, Vol.167 (7), p.1839-1852.e21 |
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| container_end_page | 1852.e21 |
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| container_title | Cell |
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| creator | Wang, Guanshi Hauver, Jesse Thomas, Zachary Darst, Seth A. Pertsinidis, Alexandros |
| description | Many essential cellular processes, such as gene control, employ elaborate mechanisms involving the coordination of large, multi-component molecular assemblies. Few structural biology tools presently have the combined spatial-temporal resolution and molecular specificity required to capture the movement, conformational changes, and subunit association-dissociation kinetics, three fundamental elements of how such intricate molecular machines work. Here, we report a 3D single-molecule super-resolution imaging study using modulation interferometry and phase-sensitive detection that achieves |
| doi_str_mv | 10.1016/j.cell.2016.11.032 |
| format | Article |
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[Display omitted]
•Real-time single-molecule tracking with <2 nm 3D localization precision•3D super-resolution imaging of molecular complexes in cells via particle averaging•Dynamic single-molecule visualization of the full E. coli RNAP transcription cycle•Dissection of promoter clearance kinetics and fate of σ70 initiation factors
Visualizing the movement of individual E. coli RNA polymerases through the complete transcription cycle is possible thanks to a novel 3D single-molecule super-resolution imaging approach.</description><identifier>ISSN: 0092-8674</identifier><identifier>EISSN: 1097-4172</identifier><identifier>DOI: 10.1016/j.cell.2016.11.032</identifier><identifier>PMID: 27984731</identifier><language>eng</language><publisher>United States: Elsevier Inc</publisher><subject>axial localization ; DNA-Directed RNA Polymerases - metabolism ; Escherichia coli - metabolism ; holoenzyme ; Humans ; Imaging, Three-Dimensional - methods ; interferometry ; Interferometry - methods ; nuclear pore complex ; RNA polymerase ; sigma factor ; Single Molecule Imaging - methods ; single-molecule ; super-resolution imaging ; transcription ; transcription cycle ; Transcription, Genetic</subject><ispartof>Cell, 2016-12, Vol.167 (7), p.1839-1852.e21</ispartof><rights>2016 Elsevier Inc.</rights><rights>Copyright © 2016 Elsevier Inc. All rights reserved.</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c455t-2bc76e59562438c7ddf2ed2a07effa8f69c2812dc1e858370d4f52080de75d253</citedby><cites>FETCH-LOGICAL-c455t-2bc76e59562438c7ddf2ed2a07effa8f69c2812dc1e858370d4f52080de75d253</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktohtml>$$Uhttps://dx.doi.org/10.1016/j.cell.2016.11.032$$EHTML$$P50$$Gelsevier$$Hfree_for_read</linktohtml><link.rule.ids>230,314,780,784,885,3548,27922,27923,45993</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/27984731$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Wang, Guanshi</creatorcontrib><creatorcontrib>Hauver, Jesse</creatorcontrib><creatorcontrib>Thomas, Zachary</creatorcontrib><creatorcontrib>Darst, Seth A.</creatorcontrib><creatorcontrib>Pertsinidis, Alexandros</creatorcontrib><title>Single-Molecule Real-Time 3D Imaging of the Transcription Cycle by Modulation Interferometry</title><title>Cell</title><addtitle>Cell</addtitle><description>Many essential cellular processes, such as gene control, employ elaborate mechanisms involving the coordination of large, multi-component molecular assemblies. Few structural biology tools presently have the combined spatial-temporal resolution and molecular specificity required to capture the movement, conformational changes, and subunit association-dissociation kinetics, three fundamental elements of how such intricate molecular machines work. Here, we report a 3D single-molecule super-resolution imaging study using modulation interferometry and phase-sensitive detection that achieves <2 nm axial localization precision, well below the few-nanometer-sized individual protein components. To illustrate the capability of this technique in probing the dynamics of complex macromolecular machines, we visualize the movement of individual multi-subunit E. coli RNA polymerases through the complete transcription cycle, dissect the kinetics of the initiation-elongation transition, and determine the fate of σ70 initiation factors during promoter escape. Modulation interferometry sets the stage for single-molecule studies of several hitherto difficult-to-investigate multi-molecular transactions that underlie genome regulation.
[Display omitted]
•Real-time single-molecule tracking with <2 nm 3D localization precision•3D super-resolution imaging of molecular complexes in cells via particle averaging•Dynamic single-molecule visualization of the full E. coli RNAP transcription cycle•Dissection of promoter clearance kinetics and fate of σ70 initiation factors
Visualizing the movement of individual E. coli RNA polymerases through the complete transcription cycle is possible thanks to a novel 3D single-molecule super-resolution imaging approach.</description><subject>axial localization</subject><subject>DNA-Directed RNA Polymerases - metabolism</subject><subject>Escherichia coli - metabolism</subject><subject>holoenzyme</subject><subject>Humans</subject><subject>Imaging, Three-Dimensional - methods</subject><subject>interferometry</subject><subject>Interferometry - methods</subject><subject>nuclear pore complex</subject><subject>RNA polymerase</subject><subject>sigma factor</subject><subject>Single Molecule Imaging - methods</subject><subject>single-molecule</subject><subject>super-resolution imaging</subject><subject>transcription</subject><subject>transcription cycle</subject><subject>Transcription, Genetic</subject><issn>0092-8674</issn><issn>1097-4172</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2016</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNp9kU9rGzEQxUVpady0X6CHssdeditppZUWSqE4_WNICKTurSBkaeTIaFeutBvwt69cpyG59DRi5vfeiHkIvSW4IZh0H3aNgRAaWt4NIQ1u6TO0ILgXNSOCPkcLjHtay06wM_Qq5x3GWHLOX6IzKnrJREsW6NcPP24D1FcxgJkDVDegQ732A1TtRbUa9LbMq-iq6RaqddJjNsnvJx_Hankwhd8cqqto56D_9lbjBMlBigNM6fAavXA6ZHhzX8_Rz69f1svv9eX1t9Xy82VtGOdTTTdGdMB73lHWSiOsdRQs1ViAc1q6rjdUEmoNAcllK7BljlMssQXBLeXtOfp08t3PmwGsgXFKOqh98oNOBxW1V08no79V23inOGOsE6QYvL83SPH3DHlSg8_H4-oR4pwVkZx2PeatLCg9oSbFnBO4hzUEq2MsaqeOSnWMRRGiSixF9O7xBx8k_3IowMcTAOVMdx6SysbDaMD6BGZSNvr_-f8B2C2f2w</recordid><startdate>20161215</startdate><enddate>20161215</enddate><creator>Wang, Guanshi</creator><creator>Hauver, Jesse</creator><creator>Thomas, Zachary</creator><creator>Darst, Seth A.</creator><creator>Pertsinidis, Alexandros</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>7X8</scope><scope>5PM</scope></search><sort><creationdate>20161215</creationdate><title>Single-Molecule Real-Time 3D Imaging of the Transcription Cycle by Modulation Interferometry</title><author>Wang, Guanshi ; Hauver, Jesse ; Thomas, Zachary ; Darst, Seth A. ; Pertsinidis, Alexandros</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c455t-2bc76e59562438c7ddf2ed2a07effa8f69c2812dc1e858370d4f52080de75d253</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2016</creationdate><topic>axial localization</topic><topic>DNA-Directed RNA Polymerases - metabolism</topic><topic>Escherichia coli - metabolism</topic><topic>holoenzyme</topic><topic>Humans</topic><topic>Imaging, Three-Dimensional - methods</topic><topic>interferometry</topic><topic>Interferometry - methods</topic><topic>nuclear pore complex</topic><topic>RNA polymerase</topic><topic>sigma factor</topic><topic>Single Molecule Imaging - methods</topic><topic>single-molecule</topic><topic>super-resolution imaging</topic><topic>transcription</topic><topic>transcription cycle</topic><topic>Transcription, Genetic</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Wang, Guanshi</creatorcontrib><creatorcontrib>Hauver, Jesse</creatorcontrib><creatorcontrib>Thomas, Zachary</creatorcontrib><creatorcontrib>Darst, Seth A.</creatorcontrib><creatorcontrib>Pertsinidis, Alexandros</creatorcontrib><collection>ScienceDirect Open Access Titles</collection><collection>Elsevier:ScienceDirect:Open Access</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>Cell</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Wang, Guanshi</au><au>Hauver, Jesse</au><au>Thomas, Zachary</au><au>Darst, Seth A.</au><au>Pertsinidis, Alexandros</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Single-Molecule Real-Time 3D Imaging of the Transcription Cycle by Modulation Interferometry</atitle><jtitle>Cell</jtitle><addtitle>Cell</addtitle><date>2016-12-15</date><risdate>2016</risdate><volume>167</volume><issue>7</issue><spage>1839</spage><epage>1852.e21</epage><pages>1839-1852.e21</pages><issn>0092-8674</issn><eissn>1097-4172</eissn><abstract>Many essential cellular processes, such as gene control, employ elaborate mechanisms involving the coordination of large, multi-component molecular assemblies. Few structural biology tools presently have the combined spatial-temporal resolution and molecular specificity required to capture the movement, conformational changes, and subunit association-dissociation kinetics, three fundamental elements of how such intricate molecular machines work. Here, we report a 3D single-molecule super-resolution imaging study using modulation interferometry and phase-sensitive detection that achieves <2 nm axial localization precision, well below the few-nanometer-sized individual protein components. To illustrate the capability of this technique in probing the dynamics of complex macromolecular machines, we visualize the movement of individual multi-subunit E. coli RNA polymerases through the complete transcription cycle, dissect the kinetics of the initiation-elongation transition, and determine the fate of σ70 initiation factors during promoter escape. Modulation interferometry sets the stage for single-molecule studies of several hitherto difficult-to-investigate multi-molecular transactions that underlie genome regulation.
[Display omitted]
•Real-time single-molecule tracking with <2 nm 3D localization precision•3D super-resolution imaging of molecular complexes in cells via particle averaging•Dynamic single-molecule visualization of the full E. coli RNAP transcription cycle•Dissection of promoter clearance kinetics and fate of σ70 initiation factors
Visualizing the movement of individual E. coli RNA polymerases through the complete transcription cycle is possible thanks to a novel 3D single-molecule super-resolution imaging approach.</abstract><cop>United States</cop><pub>Elsevier Inc</pub><pmid>27984731</pmid><doi>10.1016/j.cell.2016.11.032</doi><oa>free_for_read</oa></addata></record> |
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| source | MEDLINE; Elsevier ScienceDirect Journals Complete; Cell Press Free Archives (Open Access); EZB Electronic Journals Library |
| subjects | axial localization DNA-Directed RNA Polymerases - metabolism Escherichia coli - metabolism holoenzyme Humans Imaging, Three-Dimensional - methods interferometry Interferometry - methods nuclear pore complex RNA polymerase sigma factor Single Molecule Imaging - methods single-molecule super-resolution imaging transcription transcription cycle Transcription, Genetic |
| title | Single-Molecule Real-Time 3D Imaging of the Transcription Cycle by Modulation Interferometry |
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