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In vitro integration of ribosomal RNA synthesis, ribosome assembly, and translation
Purely in vitro ribosome synthesis could provide a critical step towards unraveling the systems biology of ribosome biogenesis, constructing minimal cells from defined components, and engineering ribosomes with new functions. Here, as an initial step towards this goal, we report a method for constru...
Autores principales: | , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
European Molecular Biology Organization
2013
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3964315/ https://www.ncbi.nlm.nih.gov/pubmed/23799452 http://dx.doi.org/10.1038/msb.2013.31 |
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author | Jewett, Michael C Fritz, Brian R Timmerman, Laura E Church, George M |
author_facet | Jewett, Michael C Fritz, Brian R Timmerman, Laura E Church, George M |
author_sort | Jewett, Michael C |
collection | PubMed |
description | Purely in vitro ribosome synthesis could provide a critical step towards unraveling the systems biology of ribosome biogenesis, constructing minimal cells from defined components, and engineering ribosomes with new functions. Here, as an initial step towards this goal, we report a method for constructing Escherichia coli ribosomes in crude S150 E. coli extracts. While conventional methods for E. coli ribosome reconstitution are non-physiological, our approach attempts to mimic chemical conditions in the cytoplasm, thus permitting several biological processes to occur simultaneously. Specifically, our integrated synthesis, assembly, and translation (iSAT) technology enables one-step co-activation of rRNA transcription, assembly of transcribed rRNA with native ribosomal proteins into functional ribosomes, and synthesis of active protein by these ribosomes in the same compartment. We show that iSAT makes possible the in vitro construction of modified ribosomes by introducing a 23S rRNA mutation that mediates resistance against clindamycin. We anticipate that iSAT will aid studies of ribosome assembly and open new avenues for making ribosomes with altered properties. |
format | Online Article Text |
id | pubmed-3964315 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2013 |
publisher | European Molecular Biology Organization |
record_format | MEDLINE/PubMed |
spelling | pubmed-39643152014-03-25 In vitro integration of ribosomal RNA synthesis, ribosome assembly, and translation Jewett, Michael C Fritz, Brian R Timmerman, Laura E Church, George M Mol Syst Biol Report Purely in vitro ribosome synthesis could provide a critical step towards unraveling the systems biology of ribosome biogenesis, constructing minimal cells from defined components, and engineering ribosomes with new functions. Here, as an initial step towards this goal, we report a method for constructing Escherichia coli ribosomes in crude S150 E. coli extracts. While conventional methods for E. coli ribosome reconstitution are non-physiological, our approach attempts to mimic chemical conditions in the cytoplasm, thus permitting several biological processes to occur simultaneously. Specifically, our integrated synthesis, assembly, and translation (iSAT) technology enables one-step co-activation of rRNA transcription, assembly of transcribed rRNA with native ribosomal proteins into functional ribosomes, and synthesis of active protein by these ribosomes in the same compartment. We show that iSAT makes possible the in vitro construction of modified ribosomes by introducing a 23S rRNA mutation that mediates resistance against clindamycin. We anticipate that iSAT will aid studies of ribosome assembly and open new avenues for making ribosomes with altered properties. European Molecular Biology Organization 2013-06-25 /pmc/articles/PMC3964315/ /pubmed/23799452 http://dx.doi.org/10.1038/msb.2013.31 Text en Copyright © 2013, EMBO and Macmillan Publishers Limited https://creativecommons.org/licenses/by-nc-sa/3.0/This article is licensed under a Creative Commons Attribution Noncommercial Share Alike 3.0 Unported Licence. To view a copy of this licence visit http://creativecommons.org/licenses/by-nc-sa/3.0/ (https://creativecommons.org/licenses/by-nc-sa/3.0/) . |
spellingShingle | Report Jewett, Michael C Fritz, Brian R Timmerman, Laura E Church, George M In vitro integration of ribosomal RNA synthesis, ribosome assembly, and translation |
title | In vitro integration of ribosomal RNA synthesis, ribosome assembly, and translation |
title_full | In vitro integration of ribosomal RNA synthesis, ribosome assembly, and translation |
title_fullStr | In vitro integration of ribosomal RNA synthesis, ribosome assembly, and translation |
title_full_unstemmed | In vitro integration of ribosomal RNA synthesis, ribosome assembly, and translation |
title_short | In vitro integration of ribosomal RNA synthesis, ribosome assembly, and translation |
title_sort | in vitro integration of ribosomal rna synthesis, ribosome assembly, and translation |
topic | Report |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3964315/ https://www.ncbi.nlm.nih.gov/pubmed/23799452 http://dx.doi.org/10.1038/msb.2013.31 |
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