Rational Re-engineering of a Transcriptional Silencing PreQ<sub>1</sub> Riboswitch

Wu, Ming‐Cheng; Lowe, Phillip T.; Robinson, Christopher J.; Vincent, Helen A.; Dixon, Neil; Leigh, J.R.; Micklefield, Jason

doi:10.1021/jacs.5b03405

Cited by 36 publications

(39 citation statements)

References 36 publications

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“…1). In all cases, repression was observed in the presence of preQ1, a finding consistent with our predictions and previous studies (6,15). Initial dose-response and kinetics experiments on riboswitches from B. subtilis and T. tencongensis indicated saturating behavior at Ն100 M preQ1 after 6 h (see Fig.…”

Section: Resultssupporting

confidence: 92%

“…When the target ligand is an endogenous metabolite, a modified riboswitch may be necessary to provide sufficient sensitivity and dynamic range to detect changes in intracellular concentration. In the case of preQ1, for example, the reporter protein ␤-galactosidase is largely repressed in the host organism B. subtilis when regulated by wild-type versus destabilized mutant riboswitches (6,15). Our T. tencongensis variants, which maintain responses over nM to M preQ1 but have greater dynamic range, may be appropriate for sensing changes in intracellular preQ1 in bacteria that synthesize queuosine.…”

Section: Discussionmentioning

confidence: 99%

“…However, because naturally occurring switches respond to endogenous metabolites, in order to serve as inducible gene regulation tools, they must be engineered to discriminate against the native metabolite and recognize instead an orthogonal, nonnatural ligand. Several studies have yielded riboswitches that bind to new ligands and retain switching behavior with moderate dynamic range (6)(7)(8)(9).…”

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confidence: 99%

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Characterization of Engineered PreQ1 Riboswitches for Inducible Gene Regulation in Mycobacteria

2017

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We report here the behavior of naturally occurring and rationally engineered preQ1 riboswitches and their application to inducible gene regulation in mycobacteria. Because mycobacteria lack preQ1 biosynthetic genes, we hypothesized that preQ1 could be used as an exogenous nonmetabolite ligand to control riboswitches in mycobacteria. Selected naturally occurring preQ1 riboswitches were assayed and successfully drove preQ1-dependent repression of a green fluorescent protein reporter in Mycobacterium smegmatis. Using structure-based design, we engineered three preQ1 riboswitches from Thermoanaerobacter tencongensis, Bacillus subtilis, and Lactobacillus rhamnosus toward achieving higher response ratios and increased repression. Assuming a steady-state model, variants of the T. tencongensis riboswitch most closely followed the predicted trends. Unexpectedly, the preQ1 dose response was best described by a model with a second, independent preQ1 binding site. This behavior was general to the preQ1 riboswitch family, since the wild type and rationally designed mutants of riboswitches from all three bacteria behaved analogously. Across all variants, the response ratios, which describe expression in the absence versus the presence of preQ1, ranged from Ͻ2 to ϳ10, but repression in all cases was incomplete up to 1 mM preQ1. By reducing the transcript expression level, we obtained a preQ1 riboswitch variant appropriate for inducible knockdown applications. We further showed that the preQ1 response is reversible, is titratable, and can be used to control protein expression in mycobacteria within infected macrophages. By engineering naturally occurring preQ1 riboswitches, we have not only extended the tools available for inducible gene regulation in mycobacteria but also uncovered new behavior of these riboswitches.IMPORTANCE Riboswitches are elements found in noncoding regions of mRNA that regulate gene expression, typically in response to an endogenous metabolite. Riboswitches have emerged as important tools for inducible gene expression in diverse organisms. We noted that mycobacteria lack the biosynthesis genes for preQ1, a ligand for riboswitches from diverse bacteria. Predicting that preQ1 is not present in mycobacteria, we showed that it controls optimized riboswitches appropriate for gene knockdown applications. Further, the riboswitch response is subject to a second independent preQ1 binding event that has not been previously documented. By engineering naturally occurring riboswitches, we have uncovered a new behavior, with implications for riboswitch function in its native context, and extended the tools available for inducible gene regulation in mycobacteria.

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Section: Resultssupporting

confidence: 92%

Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

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Characterization of Engineered PreQ1 Riboswitches for Inducible Gene Regulation in Mycobacteria

2017

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“…A modest screen of different phylogenetic and stem variants was guided by empirical design rules for functional biosensors that start with use of the natural P1 stem (Kellenberger and Hammond, 2015; Wang et al, 2016). Our results in this study again demonstrate that riboswitch-based biosensors can be reprogrammed to recognize other ligands via point mutations, although not necessarily with full selectivity (Kellenberger et al, 2013; Ren et al, 2015; Wu et al, 2015). The broader implication is that natural riboswitch-ligand pairs can serve as advanced starting points for the engineering of biosensors for related ligands not recognized by known riboswitches.…”

Section: Discussionsupporting

confidence: 58%

An RNA-Based Fluorescent Biosensor for High-Throughput Analysis of the cGAS-cGAMP-STING Pathway

Bose

Marcus

et al. 2016

Cell Chemical Biology

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Summary In mammalian cells, the second messenger (2′-5′, 3′-5′) cyclic GMP-AMP (2′, 3′-cGAMP), is produced by the cytosolic DNA sensor cGAMP synthase (cGAS), and subsequently bound by stimulator of interferon genes (STING) to trigger interferon response. Thus, the cGAS-cGAMP-STING pathway plays a critical role in pathogen detection, as well as pathophysiological conditions including cancer and autoimmune disorders. However, studying and targeting this immune signaling pathway has been challenging due to the absence of tools for high-throughput analysis. We have engineered an RNA-based fluorescent biosensor that responds to 2′, 3′-cGAMP. The resulting “mix-and-go” cGAS activity assay shows excellent statistical reliability as a high-throughput screening (HTS) assay and distinguishes between direct and indirect cGAS inhibitors. Furthermore, the biosensor enables quantitation of 2′, 3′-cGAMP in mammalian cell lysates. We envision this biosensor-based assay as a resource to study the cGAS-cGAMP-STING pathway in the context of infectious diseases, cancer immunotherapy, and autoimmune diseases.

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“…Micklefield and coworkers used a rational targeted approach in the evaluation of synthetic compounds with riboswitch mutants and identified an orthogonal riboswitch−ligand pair that effectively repressed the transcription of selected genes in B. subtilis [42]. More recently, rationally engineered preQ 1 riboswitches have been applied for inducible gene regulation in mycobacteria [43].…”

Section: Resultsmentioning

confidence: 99%

Superior cellular activities of azido- over amino-functionalized ligands for engineered preQ₁riboswitches inE.coli

Neuner¹,

Frener²,

Lusser

et al. 2018

RNA Biology

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For this study, we utilized class-I and class-II preQ1-sensing riboswitches as model systems to decipher the structure-activity relationship of rationally designed ligand derivatives in vitro and in vivo. We found that synthetic preQ1 ligands with amino-modified side chains that protrude from the ligand-encapsulating binding pocket, and thereby potentially interact with the phosphate backbone in their protonated form, retain or even increase binding affinity for the riboswitches in vitro. They, however, led to significantly lower riboswitch activities in a reporter system in vivo in E. coli. Importantly, when we substituted the amino- by azido-modified side chains, the cellular activities of the ligands were restored for the class-I conditional gene expression system and even improved for the class-II counterpart. Kinetic analysis of ligand binding in vitro revealed enhanced on-rates for amino-modified derivatives while they were attenuated for azido-modified variants. This shows that neither high affinities nor fast on-rates are necessarily translated into efficient cellular activities. Taken together, our comprehensive study interconnects in vitro kinetics and in vitro thermodynamics of RNA-ligand binding with the ligands’ in vivo performance and thereby encourages azido- rather than amino-functionalized design for enhanced cellular activity.

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Rational Re-engineering of a Transcriptional Silencing PreQ₁ Riboswitch

Cited by 36 publications

References 36 publications

Characterization of Engineered PreQ1 Riboswitches for Inducible Gene Regulation in Mycobacteria

Characterization of Engineered PreQ1 Riboswitches for Inducible Gene Regulation in Mycobacteria

An RNA-Based Fluorescent Biosensor for High-Throughput Analysis of the cGAS-cGAMP-STING Pathway

Superior cellular activities of azido- over amino-functionalized ligands for engineered preQ₁riboswitches inE.coli

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Rational Re-engineering of a Transcriptional Silencing PreQ1 Riboswitch

Cited by 36 publications

References 36 publications

Characterization of Engineered PreQ1 Riboswitches for Inducible Gene Regulation in Mycobacteria

Characterization of Engineered PreQ1 Riboswitches for Inducible Gene Regulation in Mycobacteria

An RNA-Based Fluorescent Biosensor for High-Throughput Analysis of the cGAS-cGAMP-STING Pathway

Superior cellular activities of azido- over amino-functionalized ligands for engineered preQ1riboswitches inE.coli

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Product

Resources

About

Rational Re-engineering of a Transcriptional Silencing PreQ₁ Riboswitch

Superior cellular activities of azido- over amino-functionalized ligands for engineered preQ₁riboswitches inE.coli