Rational Re-engineering of a Transcriptional Silencing PreQ1 Riboswitch.

Ming-Cheng Wu,James Leigh,Christopher J Robinson,Neil Dixon,Helen A Vincent,Jason Micklefield,Phillip T Lowe

doi:10.1021/jacs.5b03405

Ming-Cheng Wu, James Leigh + Show 5 more

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https://doi.org/10.1021/jacs.5b03405

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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.

Highlights

Most riboswitches are found within the 5′-untranslated regions (5′UTRs) of bacterial mRNAs that encode gene products required for the biosynthesis, catabolism, or transport of the cognate metabolite.[1,2]
RNA-based gene regulators, such as riboswitches, are an attractive starting point for the development of new gene expression tools for the synthetic biology and biotechnology fields.[4−8] RNA regulatory components have the advantage over their protein counterparts in that they are inherently more versatile, being relatively easy to design or manipulate in a predictable manner and being transferable between organisms.[4−6] Natural riboswitches provide a diverse array of readymade components, with different aptamer domains having been identified for more than 20 specific metabolite ligands[1,2] and expression platforms that exhibit considerable mechanistic diversity.[1,2]
The most common strategy used to create such switches is to insert RNA aptamers that have been generated by in vitro selection into the 5′-UTR of a target gene and to use a genetic selection or screen to isolate functioning riboswitches.[10−13] Recent studies have shown that hybrid riboswitches can be rationally designed, through the fusion of synthetic aptamers with expression platforms derived from natural riboswitches, to create functional chimeric switches.[14,15]

Summary

Introduction

Riboswitches are noncoding regions of mRNA that regulate gene expression through the selective binding of metabolites.[1,2] Most riboswitches are found within the 5′-untranslated regions (5′UTRs) of bacterial mRNAs that encode gene products required for the biosynthesis, catabolism, or transport of the cognate metabolite.[1,2] Riboswitches exhibit a modular architecture consisting of an aptamer domain that, upon ligand binding, induces a conformational change in an overlapping expression platform, modulating gene expression, primarily through transcription termination or translation initiation.[1−3]RNA-based gene regulators, such as riboswitches, are an attractive starting point for the development of new gene expression tools for the synthetic biology and biotechnology fields.[4−8] RNA regulatory components have the advantage over their protein counterparts in that they are inherently more versatile, being relatively easy to design or manipulate in a predictable manner and being transferable between organisms.[4−6] Natural riboswitches provide a diverse array of readymade components, with different aptamer domains having been identified for more than 20 specific metabolite ligands[1,2] and expression platforms that exhibit considerable mechanistic diversity.[1,2] The modularity of these regulatory components is demonstrated by the natural occurrence of tandem riboswitches, which can function as two-input Boolean logic gates or provide more binary switching responses.[2]. Engineered riboswitches that respond to synthetic non-natural ligands, rather than metabolites, are highly desirable.[4] The most common strategy used to create such switches is to insert RNA aptamers that have been generated by in vitro selection into the 5′-UTR of a target gene and to use a genetic selection or screen to isolate functioning riboswitches.[10−13] Recent studies have shown that hybrid riboswitches can be rationally designed, through the fusion of synthetic aptamers with expression platforms derived from natural riboswitches, to create functional chimeric switches.[14,15] In theory, it should be possible to generate a riboswitch that responds to any ligand of choice using these approaches. This is because the development of new aptamers in vitro can be laborious and, critically, does not guarantee high specificity or functionality in vivo.[4,17]

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