RNA aptamers as genetic control devices: The potential of riboswitches as synthetic elements for regulating gene expression

Berens, Christian; Groher, Florian; Suess, Beatrix

doi:10.1002/biot.201300498

Cited by 95 publications

(82 citation statements)

References 135 publications

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“…Numerous artificial aptamers have been generated by SELEX over the past decades for synthetic biology and therapeutic needs (Berens et al, 2015; Soldevilla et al, 2016). In contrast, natural RNA aptamers, such as riboswitches, have been already utilized by living cells to sense and efficiently respond to their environment (Breaker, 2012; Mellin and Cossart, 2015).…”

Section: Discussionmentioning

confidence: 99%

Natural RNA Polymerase Aptamers Regulate Transcription in E. coli

et al. 2017

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Summary In search for RNA signals that modulate transcription via direct interaction with RNA polymerase (RNAP) we deep-sequenced an E. coli genomic library enriched for RNAP-binding RNAs. Many natural RNAP-binding aptamers, termed RAPs, were mapped to the genome. Over 60% of E. coli genes carry RAPs in their mRNA. Combining in vitro and in vivo approaches we characterized a subset of RAPs (iRAPs) that promote Rho-dependent transcription termination. A representative iRAP within the coding region of the essential gene, nadD, greatly reduces its transcriptional output in stationary phase and under oxidative stress, demonstrating that iRAPs control gene expression in response to changing environment. The mechanism of iRAPs involves active uncoupling of transcription and translation, making nascent RNA accessible to Rho. iRAPs encoded in the antisense strand also promote gene expression by reducing transcriptional interference. In essence, our work uncovers a broad class of cis-acting RNA signals that globally control bacterial transcription.

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Section: Discussionmentioning

confidence: 99%

Natural RNA Polymerase Aptamers Regulate Transcription in E. coli

et al. 2017

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“…5,6 Examples of such artificial systems are allosteric ribozymes and synthetic riboswitches that are used for biosensors and gene regulation systems. 7 A useful characteristic of these systems is that the intrinsic aptamer domain, which is a recognition unit for the target molecule, can be converted to another aptamer domain to alter target molecule specificity. 8 Artificial RNA functional switches that respond to various biologically relevant molecules have been developed by using aptamer domains obtained by a synthetic evolution of ligands by exponential enrichment (SELEX) technology.…”

Section: * S Supporting Informationmentioning

confidence: 99%

Rational Design and Tuning of Functional RNA Switch to Control an Allosteric Intermolecular Interaction

Endoh

Sugimoto

2015

Anal. Chem.

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Conformational transitions of biomolecules in response to specific stimuli control many biological processes. In natural functional RNA switches, often called riboswitches, a particular RNA structure that has a suppressive or facilitative effect on gene expression transitions to an alternative structure with the opposite effect upon binding of a specific metabolite to the aptamer region. Stability of RNA secondary structure (-ΔG°) can be predicted based on thermodynamic parameters and is easily tuned by changes in nucleobases. We envisioned that tuning of a functional RNA switch that causes an allosteric interaction between an RNA and a peptide would be possible based on a predicted switching energy (ΔΔG°) that corresponds to the energy difference between the RNA secondary structure before (-ΔG°before) and after (-ΔG°after) the RNA conformational transition. We first selected functional RNA switches responsive to neomycin with predicted ΔΔG° values ranging from 5.6 to 12.2 kcal mol(-1). We then demonstrated a simple strategy to rationally convert the functional RNA switch to switches responsive to natural metabolites thiamine pyrophosphate, S-adenosyl methionine, and adenine based on the predicted ΔΔG° values. The ΔΔG° values of the designed RNA switches proportionally correlated with interaction energy (ΔG°interaction) between the RNA and peptide, and we were able to tune the sensitivity of the RNA switches for the trigger molecule. The strategy demonstrated here will be generally applicable for construction of functional RNA switches and biosensors in which mechanisms are based on conformational transition of nucleic acids.

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“…Therefore, it is worthwhile to assess the performance of riboswitch elements in the context of individual genetic backgrounds. Usually, several features such as regulatory efficiency, basal activity, noise, and kinetic parameters are systematically considered under similar experimental settings [24]. The continuous increase in the induction ratios over activation at different times suggested that the induction by P43′-riboE1 functions regularly in the B. subtilis host.…”

Section: Discussionmentioning

confidence: 99%

Engineering an inducible gene expression system for Bacillus subtilis from a strong constitutive promoter and a theophylline-activated synthetic riboswitch

et al. 2016

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BackgroundSynthetic riboswitches have been increasingly used to control and tune gene expression in diverse organisms. Although a set of theophylline-responsive riboswitches have been developed for bacteria, fully functional expression elements mediated by synthetic riboswitches in Bacillus subtilis are rarely used because of the host-dependent compatibility between the promoters and riboswitches.ResultsA novel genetic element composed of the promoter P43 and a theophylline-riboswitch was developed and characterized in B. subtilis. When combined with a P43 promoter (P43′-riboE1), the theophylline-riboswitch successfully switched the constitutive expression pattern of P43 to an induced pattern. The expression mediated by the novel element could be activated at the translational level by theophylline with a relatively high induction ratio. The induction ratios for P43′-riboE1 by 4-mM theophylline were elevated during the induction period. The level of induced expression was dependent on the theophylline dose. Correspondingly, the induction ratios gradually increased in parallel with the elevated dose of theophylline. Importantly, the induced expression level was higher than three other strong constitutive promoters including PsrfA, PaprE, and the native P43. It was found that the distance between the SD sequence within the expression element and the start codon significantly influenced both the level of induced expression and the induction ratio. A 9-bp spacer was suitable for producing desirable expression level and induction ratio. Longer spacer reduced the activation efficiency. Importantly, the system successfully overexpressed β-glucuronidase at equal levels, and induction ratio was similar to that of GFP.ConclusionThe constructed theophylline-inducible gene expression system has broad compatibility and robustness, which has great potential in over-production of pharmaceutical and industrial proteins and utilization in building more complex gene circuits.

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RNA aptamers as genetic control devices: The potential of riboswitches as synthetic elements for regulating gene expression

Cited by 95 publications

References 135 publications

Natural RNA Polymerase Aptamers Regulate Transcription in E. coli

Natural RNA Polymerase Aptamers Regulate Transcription in E. coli

Rational Design and Tuning of Functional RNA Switch to Control an Allosteric Intermolecular Interaction

Engineering an inducible gene expression system for Bacillus subtilis from a strong constitutive promoter and a theophylline-activated synthetic riboswitch

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