Transcribing Genes the Hard Way: In Vitro Reconstitution of Nanoarchaeal RNA Polymerase Reveals Unusual Active Site Properties
Nanoarchaea represent a highly diverged archaeal phylum that displays many unusual biological features. The Nanoarchaeum equitans genome encodes a complete set of RNA polymerase (RNAP) subunits and basal factors. Several of the standard motifs in the active center contain radical substitutions that...
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| Format: | Article |
| Language: | English |
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Frontiers Media S.A.
2021-05-01
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| Series: | Frontiers in Molecular Biosciences |
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| Online Access: | https://www.frontiersin.org/articles/10.3389/fmolb.2021.669314/full |
| _version_ | 1797666427606401024 |
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| author | Sven Nottebaum Sven Nottebaum Robert O. J. Weinzierl |
| author_facet | Sven Nottebaum Sven Nottebaum Robert O. J. Weinzierl |
| author_sort | Sven Nottebaum |
| collection | DOAJ |
| description | Nanoarchaea represent a highly diverged archaeal phylum that displays many unusual biological features. The Nanoarchaeum equitans genome encodes a complete set of RNA polymerase (RNAP) subunits and basal factors. Several of the standard motifs in the active center contain radical substitutions that are normally expected to render the polymerase catalytically inactive. Here we show that, despite these unusual features, a RNAP reconstituted from recombinant Nanoarchaeum subunits is transcriptionally active. Using a sparse-matrix high-throughput screening method we identified an atypical stringent requirement for fluoride ions to maximize its activity under in vitro transcription conditions. |
| first_indexed | 2024-03-11T19:59:15Z |
| format | Article |
| id | doaj.art-35aceeb8d0e94eb3b97dc2b7d8de1cc2 |
| institution | Directory Open Access Journal |
| issn | 2296-889X |
| language | English |
| last_indexed | 2024-03-11T19:59:15Z |
| publishDate | 2021-05-01 |
| publisher | Frontiers Media S.A. |
| record_format | Article |
| series | Frontiers in Molecular Biosciences |
| spelling | doaj.art-35aceeb8d0e94eb3b97dc2b7d8de1cc22023-10-04T11:30:54ZengFrontiers Media S.A.Frontiers in Molecular Biosciences2296-889X2021-05-01810.3389/fmolb.2021.669314669314Transcribing Genes the Hard Way: In Vitro Reconstitution of Nanoarchaeal RNA Polymerase Reveals Unusual Active Site PropertiesSven Nottebaum0Sven Nottebaum1Robert O. J. Weinzierl2Department of Life Sciences, Imperial College London, London, United KingdomOrthomol Pharmazeutische Vertriebs GmbH, Langenfeld, GermanyDepartment of Life Sciences, Imperial College London, London, United KingdomNanoarchaea represent a highly diverged archaeal phylum that displays many unusual biological features. The Nanoarchaeum equitans genome encodes a complete set of RNA polymerase (RNAP) subunits and basal factors. Several of the standard motifs in the active center contain radical substitutions that are normally expected to render the polymerase catalytically inactive. Here we show that, despite these unusual features, a RNAP reconstituted from recombinant Nanoarchaeum subunits is transcriptionally active. Using a sparse-matrix high-throughput screening method we identified an atypical stringent requirement for fluoride ions to maximize its activity under in vitro transcription conditions.https://www.frontiersin.org/articles/10.3389/fmolb.2021.669314/fullarchaeananoarchaeaRNA polymerasecatalytic centeractive sitehigh-throughput assay |
| spellingShingle | Sven Nottebaum Sven Nottebaum Robert O. J. Weinzierl Transcribing Genes the Hard Way: In Vitro Reconstitution of Nanoarchaeal RNA Polymerase Reveals Unusual Active Site Properties Frontiers in Molecular Biosciences archaea nanoarchaea RNA polymerase catalytic center active site high-throughput assay |
| title | Transcribing Genes the Hard Way: In Vitro Reconstitution of Nanoarchaeal RNA Polymerase Reveals Unusual Active Site Properties |
| title_full | Transcribing Genes the Hard Way: In Vitro Reconstitution of Nanoarchaeal RNA Polymerase Reveals Unusual Active Site Properties |
| title_fullStr | Transcribing Genes the Hard Way: In Vitro Reconstitution of Nanoarchaeal RNA Polymerase Reveals Unusual Active Site Properties |
| title_full_unstemmed | Transcribing Genes the Hard Way: In Vitro Reconstitution of Nanoarchaeal RNA Polymerase Reveals Unusual Active Site Properties |
| title_short | Transcribing Genes the Hard Way: In Vitro Reconstitution of Nanoarchaeal RNA Polymerase Reveals Unusual Active Site Properties |
| title_sort | transcribing genes the hard way in vitro reconstitution of nanoarchaeal rna polymerase reveals unusual active site properties |
| topic | archaea nanoarchaea RNA polymerase catalytic center active site high-throughput assay |
| url | https://www.frontiersin.org/articles/10.3389/fmolb.2021.669314/full |
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