Tuning Riboswitch‐Mediated Gene Regulation by Rational Control of Aptamer Ligand Binding Properties

Rode, Ambadas B.; Endoh, Tamaki; Sugimoto, Naoki

doi:10.1002/anie.201407385

Cited by 36 publications

(28 citation statements)

References 29 publications

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“…Thea ssociation (k on )a nd dissociation (k off )r ate constants are shown in Table 1. [23] Theo bserved decrease in k on (ca. [23] Theo bserved decrease in k on (ca.…”

mentioning

confidence: 99%

Crowding Shifts the FMN Recognition Mechanism of Riboswitch Aptamer from Conformational Selection to Induced Fit

Rode¹,

Endoh²,

Sugimoto

2018

Angewandte Chemie

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In bacteria, the binding between the riboswitch aptamer domain and ligand is regulated by environmental cues,such as lowMg 2+ in macrophages during pathogenesis to ensure spatiotemporal expression of virulence genes.B inding was investigated between the flavin mononucleotide (FMN) riboswitch aptamer and its anionic ligand in the presence of molecular crowding agent without Mg 2+ ion, whichm imics pathogenic conditions.Structural, kinetic, and thermodynamic analyses under the crowding revealed more dynamic conformational rearrangements of the FMN riboswitch aptamer compared to dilute Mg 2+ -containing solution. It is hypothesized that under crowding conditions FMN binds through an induced fit mechanism in contrast to the conformational selection mechanism previously demonstrated in dilute Mg 2+ solution. Since these two mechanisms involve different conformational intermediates and rate constants,these findings have practical significance in areas such as drug design and RNAengineering.Bacteria must survive in various extracellular and intracellular environments during infection. [1] Fore xample,t he divalent magnesium cation (Mg 2+ )c oncentration is significantly lower during intramacrophage survival and growth of bacteria than it is during in vitro culture. [2] Bacteria modulate expression of certain genes to survive and replicate in low Mg 2+ environments. [2,3] Bacteria sense the decrease in Mg 2+ levels to achieve temporal and spatial expression of virulence genes. [4] TheMg 2+ ion is crucial for RNAfunctions as it neutralizes the phosphate backbone to stabilize RNAt ertiary structure formation. [5] Thus,t he fluctuation of intracellular Mg 2+ concentration during bacterial pathogenesis will have significant impacts on regulatory RNAc onformations and functions.Indeed, Mg 2+ -sensing riboswitches modulate expression of genes responsible for magnesium transport across the membrane as well as of genes involved in virulence. [2,6] Although Mg 2+ -sensing riboswitches are well-studied, [6] the effects of aM g 2+ -depleted environment on metabolite-sens-ing riboswitches, [7] which regulate the genes essential for survival and growth during bacterial pathogenesis,a re not characterized. Metabolite-sensing riboswitches depend on Mg 2+ to stabilize tertiary structures indispensable for function. [5] Affinities of riboswitches for their anionic ligands such as thiamine pyrophosphate, [8] glucosamine-6-phosphate, [9] and flavin mono nucleotide (FMN) [10] are decreased by up to 1000-fold upon disruption of the Mg 2+ -mediated interactions.S ince metabolite-sensing riboswitches are promising antibacterial drug targets, [11] understanding how riboswitchligand interactions respond to Mg 2+ concentration fluctuations is crucial for development of antibacterial drugs.Most structural and ligand binding studies of riboswitches have been performed in dilute solutions [12] that substantially differ from the intracellular environment in which riboswitches function. [13] Living cells contain between 20 and 40 wt % protein, nucleic...

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“…Thea ssociation (k on )a nd dissociation (k off )r ate constants are shown in Table 1. [23] Theo bserved decrease in k on (ca. [23] Theo bserved decrease in k on (ca.…”

mentioning

confidence: 99%

Crowding Shifts the FMN Recognition Mechanism of Riboswitch Aptamer from Conformational Selection to Induced Fit

Rode¹,

Endoh²,

Sugimoto

2018

Angewandte Chemie

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“…One potential strategy for more conservative mutations is to examine sequence variation in the expression platform across all riboswitches of a given class and type. This "evolutionary" approach has been applied to the FMN riboswitch to characterize how modulation of the P1 stem of the aptamer alters the ligand on and off rates and hence the dissociation constant, although only binding and not FMN-responsive function was assayed in this in vitro study (29).…”

Section: Discussionmentioning

confidence: 99%

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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“…Many research laboratories have already obtained ACEMBL reagents, and ACEMBL systems are in the process of being integrated into structural genomics pipelines. We expect in the coming years numerous more exploits brought about by our multigene delivery technologies, and we anticipate productive synergies with other multigene recombineering tools, to deconvolute internal redundancy and explore functional structure in complex biological systems [66][67][68][69].…”

Section: 1/ Acembling Dna For Structural and Molecular Biologymentioning

confidence: 99%

ACEMBL Tool-Kits for High-Throughput Multigene Delivery and Expression in Prokaryotic and Eukaryotic Hosts

Nie

Chaillet

Becke

et al. 2016

Advances in Experimental Medicine and Biology

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Tuning Riboswitch‐Mediated Gene Regulation by Rational Control of Aptamer Ligand Binding Properties

Cited by 36 publications

References 29 publications

Crowding Shifts the FMN Recognition Mechanism of Riboswitch Aptamer from Conformational Selection to Induced Fit

Crowding Shifts the FMN Recognition Mechanism of Riboswitch Aptamer from Conformational Selection to Induced Fit

Characterization of Engineered PreQ1 Riboswitches for Inducible Gene Regulation in Mycobacteria

ACEMBL Tool-Kits for High-Throughput Multigene Delivery and Expression in Prokaryotic and Eukaryotic Hosts

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