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Achieving large dynamic range control of gene expression with a compact RNA transcription–translation regulator

RNA transcriptional regulators are emerging as versatile components for genetic network construction. However, these regulators suffer from incomplete repression in their OFF state, making their dynamic range less than that of their protein counterparts. This incomplete repression causes expression...

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Detalles Bibliográficos
Autores principales: Westbrook, Alexandra M., Lucks, Julius B.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Oxford University Press 2017
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5435934/
https://www.ncbi.nlm.nih.gov/pubmed/28387839
http://dx.doi.org/10.1093/nar/gkx215
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author Westbrook, Alexandra M.
Lucks, Julius B.
author_facet Westbrook, Alexandra M.
Lucks, Julius B.
author_sort Westbrook, Alexandra M.
collection PubMed
description RNA transcriptional regulators are emerging as versatile components for genetic network construction. However, these regulators suffer from incomplete repression in their OFF state, making their dynamic range less than that of their protein counterparts. This incomplete repression causes expression leak, which impedes the construction of larger synthetic regulatory networks as leak propagation can interfere with desired network function. To address this, we demonstrate how naturally derived antisense RNA-mediated transcriptional regulators can be configured to regulate both transcription and translation in a single compact RNA mechanism that functions in Escherichia coli. Using in vivo gene expression assays, we show that a combination of transcriptional termination and ribosome binding site sequestration increases repression from 85% to 98%, or activation from 10-fold to over 900-fold, in response to cognate antisense RNAs. We also show that orthogonal repressive versions of this mechanism can be created through engineering minimal antisense RNAs. Finally, to demonstrate the utility of this mechanism, we use it to reduce network leak in an RNA-only cascade. We anticipate these regulators will find broad use as synthetic biology moves beyond parts engineering to the design and construction of more sophisticated regulatory networks.
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spelling pubmed-54359342017-05-22 Achieving large dynamic range control of gene expression with a compact RNA transcription–translation regulator Westbrook, Alexandra M. Lucks, Julius B. Nucleic Acids Res Synthetic Biology and Bioengineering RNA transcriptional regulators are emerging as versatile components for genetic network construction. However, these regulators suffer from incomplete repression in their OFF state, making their dynamic range less than that of their protein counterparts. This incomplete repression causes expression leak, which impedes the construction of larger synthetic regulatory networks as leak propagation can interfere with desired network function. To address this, we demonstrate how naturally derived antisense RNA-mediated transcriptional regulators can be configured to regulate both transcription and translation in a single compact RNA mechanism that functions in Escherichia coli. Using in vivo gene expression assays, we show that a combination of transcriptional termination and ribosome binding site sequestration increases repression from 85% to 98%, or activation from 10-fold to over 900-fold, in response to cognate antisense RNAs. We also show that orthogonal repressive versions of this mechanism can be created through engineering minimal antisense RNAs. Finally, to demonstrate the utility of this mechanism, we use it to reduce network leak in an RNA-only cascade. We anticipate these regulators will find broad use as synthetic biology moves beyond parts engineering to the design and construction of more sophisticated regulatory networks. Oxford University Press 2017-05-19 2017-04-06 /pmc/articles/PMC5435934/ /pubmed/28387839 http://dx.doi.org/10.1093/nar/gkx215 Text en © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research. http://creativecommons.org/licenses/by/4.0/ This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.
spellingShingle Synthetic Biology and Bioengineering
Westbrook, Alexandra M.
Lucks, Julius B.
Achieving large dynamic range control of gene expression with a compact RNA transcription–translation regulator
title Achieving large dynamic range control of gene expression with a compact RNA transcription–translation regulator
title_full Achieving large dynamic range control of gene expression with a compact RNA transcription–translation regulator
title_fullStr Achieving large dynamic range control of gene expression with a compact RNA transcription–translation regulator
title_full_unstemmed Achieving large dynamic range control of gene expression with a compact RNA transcription–translation regulator
title_short Achieving large dynamic range control of gene expression with a compact RNA transcription–translation regulator
title_sort achieving large dynamic range control of gene expression with a compact rna transcription–translation regulator
topic Synthetic Biology and Bioengineering
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5435934/
https://www.ncbi.nlm.nih.gov/pubmed/28387839
http://dx.doi.org/10.1093/nar/gkx215
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