title: Rational Re-engineering of a Transcriptional Silencing PreQ1 Riboswitch.
abstract: 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.
authors.affiliation: School of Chemistry and Manchester Institute of Biotechnology, The University of Manchester, 131 Princess Street, Manchester M1 7DN, United Kingdom.
authors.forename: Ming-Cheng
authors.initials: MC
authors.lastname: Wu
authors.affiliation: School of Chemistry and Manchester Institute of Biotechnology, The University of Manchester, 131 Princess Street, Manchester M1 7DN, United Kingdom.
authors.forename: Phillip T
authors.initials: PT
authors.lastname: Lowe
authors.affiliation: School of Chemistry and Manchester Institute of Biotechnology, The University of Manchester, 131 Princess Street, Manchester M1 7DN, United Kingdom.
authors.forename: Christopher J
authors.initials: CJ
authors.lastname: Robinson
authors.affiliation: School of Chemistry and Manchester Institute of Biotechnology, The University of Manchester, 131 Princess Street, Manchester M1 7DN, United Kingdom.
authors.forename: Helen A
authors.initials: HA
authors.lastname: Vincent
authors.affiliation: School of Chemistry and Manchester Institute of Biotechnology, The University of Manchester, 131 Princess Street, Manchester M1 7DN, United Kingdom.
authors.forename: Neil
authors.initials: N
authors.lastname: Dixon
authors.affiliation: School of Chemistry and Manchester Institute of Biotechnology, The University of Manchester, 131 Princess Street, Manchester M1 7DN, United Kingdom.
authors.forename: James
authors.initials: J
authors.lastname: Leigh
authors.affiliation: School of Chemistry and Manchester Institute of Biotechnology, The University of Manchester, 131 Princess Street, Manchester M1 7DN, United Kingdom.
authors.forename: Jason
authors.initials: J
authors.lastname: Micklefield
mesh_terms: D052157:Aptamers, Nucleotide / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D001412:Bacillus subtilis / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D001483:Base Sequence; D015964:Gene Expression Regulation, Bacterial; D020868:Gene Silencing; D005818:Genetic Engineering; D016296:Mutagenesis; D011744:Pyrimidinones / Q000737:chemistry / Q000378:metabolism*; D011758:Pyrroles / Q000737:chemistry / Q000378:metabolism*; D058928:Riboswitch; D058615:Synthetic Biology; D015533:Transcriptional Activation
chemical_list: C096876:7-(aminomethyl)-7-deazaguanine; D052157:Aptamers, Nucleotide; D011744:Pyrimidinones; D011758:Pyrroles; D058928:Riboswitch