Rational re-engineering of a transcriptional silencing PreQ1 riboswitch
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Re-engineered riboswitches that no longer respond to cellular metabolites, but that instead can be controlled by synthetic molecules, are potentially useful gene regulatory tools for use in synthetic biology and biotechnology fields. Previously, extensive genetic selection and screening approaches were employed to re-engineer a natural adenine riboswitch to create orthogonal ON-switches, enabling translational control of target gene expression in response to synthetic ligands. Here, we describe how a rational targeted approach was used to re-engineer the PreQ1 riboswitch from Bacillus subtilis into an orthogonal OFF-switch. In this case, the evaluation of just six synthetic compounds with seven riboswitch mutants led to the identification of an orthogonal riboswitch-ligand pairing that effectively repressed the transcription of selected genes in B. subtilis. The streamlining of the re-engineering approach, and its extension to a second class of riboswitches, provides a methodological platform for the creation of new orthogonal regulatory components for biotechnological applications including gene functional analysis and antimicrobial target validation and screening. (Graph Presented).
Wu , M-C , Lowe , P T , Robinson , C J , Vincent , H A , Dixon , N , Leigh , J & Micklefield , J 2015 , ' Rational re-engineering of a transcriptional silencing PreQ1 riboswitch ' Journal of the American Chemical Society , vol 137 , no. 28 , pp. 9015-9021 . DOI: 10.1021/jacs.5b03405
Journal of the American Chemical Society
Copyright © 2015 American Chemical Society. This is an open access article published under a Creative Commons Attribution (CC-BY) License, which permits unrestricted use, distribution and reproduction in any medium, provided the author and source are cited.
DescriptionThis work was supported by BBSRC grant BB/I012648/1, Manchester Centre for Synthetic Biology (SynBioChem) BB/M017702/1, and a BBSRC Ph.D. studentship (for J.L.). The EPSRC provided a Ph.D. studentship (for P.T.L.).
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