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Ronald R Breaker - One of the best experts on this subject based on the ideXlab platform.
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biochemical validation of a second class of tetrahydrofolate Riboswitches in bacteria
RNA, 2019Co-Authors: Xi Chen, Gayan Mirihana Arachchilage, Ronald R BreakerAbstract:: We previously reported a large collection of structured noncoding RNAs (ncRNAs) that includes many Riboswitch candidates identified through comparative sequence analysis of bacterial intergenic regions. One of these candidates, initially named the "folE motif," adopts a simple architecture commonly found upstream of folE genes. FolE enzymes catalyze the first enzyme in the de novo folate biosynthesis pathway. Herein, we demonstrate that folE motif RNAs selectively bind the enzyme cofactor tetrahydrofolate (THF) and several of its close derivatives. These aptamers, commonly found in Gram-negative bacteria, are distinct from aptamers of the previous validated THF Riboswitch class found in Gram-positive bacteria. Our findings indicate that folE motif RNAs are aptamer domains for a second THF Riboswitch class, named THF-II. The biochemical validation of THF-II Riboswitches further highlights the ability of bacteria to utilize diverse RNA structures to sense universal enzyme cofactors that are predicted to be of ancient origin.
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SAM-VI RNAs selectively bind S-adenosylmethionine and exhibit similarities to SAM-III Riboswitches
RNA Biology, 2018Co-Authors: Gayan Mirihana Arachchilage, Zasha Weinberg, Madeline E. Sherlock, Ronald R BreakerAbstract:ABSTRACTFive distinct Riboswitch classes that regulate gene expression in response to the cofactor S-adenosylmethionine (SAM) or its metabolic breakdown product S-adenosylhomocysteine (SAH) have been reported previously. Collectively, these SAM- or SAH-sensing RNAs constitute the most abundant collection of Riboswitches, and are found in nearly every major bacterial lineage. Here, we report a potential sixth member of this pervasive Riboswitch family, called SAM-VI, which is predominantly found in Bifidobacterium species. SAM-VI aptamers selectively bind the cofactor SAM and strongly discriminate against SAH. The consensus sequence and structural model for SAM-VI share some features with the consensus model for the SAM-III Riboswitch class, whose members are mainly found in lactic acid bacteria. However, there are sufficient differences between the two classes such that current bioinformatics methods separately cluster representatives of the two motifs. These findings highlight the abundance of RNA struct...
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Riboswitch diversity and distribution
RNA, 2017Co-Authors: Phillip J. Mccown, Madeline E. Sherlock, Ronald R Breaker, Keith A Corbino, Shira StavAbstract:: Riboswitches are commonly used by bacteria to detect a variety of metabolites and ions to regulate gene expression. To date, nearly 40 different classes of Riboswitches have been discovered, experimentally validated, and modeled at atomic resolution in complex with their cognate ligands. The research findings produced since the first Riboswitch validation reports in 2002 reveal that these noncoding RNA domains exploit many different structural features to create binding pockets that are extremely selective for their target ligands. Some Riboswitch classes are very common and are present in bacteria from nearly all lineages, whereas others are exceedingly rare and appear in only a few species whose DNA has been sequenced. Presented herein are the consensus sequences, structural models, and phylogenetic distributions for all validated Riboswitch classes. Based on our findings, we predict that there are potentially many thousands of distinct bacterial Riboswitch classes remaining to be discovered, but that the rarity of individual undiscovered classes will make it increasingly difficult to find additional examples of this RNA-based sensory and gene control mechanism.
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bioinformatic analysis of Riboswitch structures uncovers variant classes with altered ligand specificity
Proceedings of the National Academy of Sciences of the United States of America, 2017Co-Authors: Zasha Weinberg, Madeline E. Sherlock, Ronald R Breaker, Christina E. Lünse, James W NelsonAbstract:Riboswitches are RNAs that form complex, folded structures that selectively bind small molecules or ions. As with certain groups of protein enzymes and receptors, some Riboswitch classes have evolved to change their ligand specificity. We developed a procedure to systematically analyze known Riboswitch classes to find additional variants that have altered their ligand specificity. This approach uses multiple-sequence alignments, atomic-resolution structural information, and Riboswitch gene associations. Among the discoveries are unique variants of the guanine Riboswitch class that most tightly bind the nucleoside 2′-deoxyguanosine. In addition, we identified variants of the glycine Riboswitch class that no longer recognize this amino acid, additional members of a rare flavin mononucleotide (FMN) variant class, and also variants of c-di-GMP-I and -II Riboswitches that might recognize different bacterial signaling molecules. These findings further reveal the diverse molecular sensing capabilities of RNA, which highlights the potential for discovering a large number of additional natural Riboswitch classes.
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Biochemical Validation of a Third Guanidine Riboswitch Class in Bacteria.
Biochemistry, 2017Co-Authors: Madeline E. Sherlock, Ronald R BreakerAbstract:Recently, it was determined that representatives of the Riboswitch candidates called ykkC and mini-ykkC directly bind free guanidine. These Riboswitches regulate the expression of genes whose protein products are implicated in overcoming the toxic effects of this ligand. Thus, the relevant ykkC motif and mini-ykkC motif RNAs have been classified as guanidine-I and guanidine-II Riboswitch RNAs, respectively. Moreover, we had previously noted that a third candidate Riboswitch class, called ykkC-III, was associated with a distribution of genes similar to those of the other two motifs. Therefore, it was predicted that ykkC-III motif RNAs would sense and respond to the same ligand. In this report, we present biochemical data supporting the hypothesis that ykkC-III RNAs represent a third class of guanidine-sensing RNAs called guanidine-III Riboswitches. Members of the guanidine-III Riboswitch class bind their ligand with an affinity similar to that observed for members of the other two classes. Notably, there are some sequence similarities between guanidine-II and guanidine-III Riboswitches. However, the characteristics of ligand discrimination by guanidine-III RNAs are different from those of the other guanidine-binding motifs, suggesting that the binding pockets have distinct features among the three Riboswitch classes.
Autumn Estrada - One of the best experts on this subject based on the ideXlab platform.
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Attempted use of PACE for Riboswitch discovery generates three new translational theophylline Riboswitch side products.
BMC Research Notes, 2018Co-Authors: Zachary M. Shaver, Stephanie S. Bent, Steven R. Bilby, Itzayana G. Cuellar, Athena J. Davis, Lindsay Doolan, Fatima C. Enriquez, Andreas Buser, Michael Brown, Autumn EstradaAbstract:The purpose of this project was to use an in vivo method to discover Riboswitches that are activated by new ligands. We employed phage-assisted continuous evolution (PACE) to evolve new Riboswitches in vivo. We started with one translational Riboswitch and one transcriptional Riboswitch, both of which were activated by theophylline. We used xanthine as the new target ligand during positive selection followed by negative selection using theophylline. The goal was to generate very large M13 phage populations that contained unknown mutations, some of which would result in new aptamer specificity. We discovered side products of three new theophylline translational Riboswitches with different levels of protein production. We used next generation sequencing to identify M13 phage that carried Riboswitch mutations. We cloned and characterized the most abundant Riboswitch mutants and discovered three variants that produce different levels of translational output while retaining their theophylline specificity. Although we were unable to demonstrate evolution of new Riboswitch ligand specificity using PACE, we recommend careful design of recombinant M13 phage to avoid evolution of “cheaters” that short circuit the intended selection pressure.
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Attempted use of PACE for Riboswitch discovery generates three new translational theophylline Riboswitch side products 06 Biological Sciences 0604 Genetics
BMC Research Notes, 2018Co-Authors: Zachary M. Shaver, Stephanie S. Bent, Steven R. Bilby, Itzayana G. Cuellar, Athena J. Davis, Lindsay Doolan, Fatima C. Enriquez, Andreas Buser, Michael Brown, Autumn EstradaAbstract:The purpose of this project was to use an in vivo method to discover Riboswitches that are activated by new ligands. We employed phage-assisted continuous evolution (PACE) to evolve new Riboswitches in vivo. We started with one translational Riboswitch and one transcriptional Riboswitch, both of which were activated by theophylline. We used xanthine as the new target ligand during positive selection followed by negative selection using theophylline. The goal was to generate very large M13 phage populations that contained unknown mutations, some of which would result in new aptamer specificity. We discovered side products of three new theophylline translational Riboswitches with different levels of protein production. We used next generation sequencing to identify M13 phage that carried Riboswitch mutations. We cloned and characterized the most abundant Riboswitch mutants and discovered three variants that produce different levels of translational output while retaining their theophylline specificity. Although we were unable to demonstrate evolution of new Riboswitch ligand specificity using PACE, we recommend careful design of recombinant M13 phage to avoid evolution of “cheaters” that short circuit the intended selection pressure.
Zasha Weinberg - One of the best experts on this subject based on the ideXlab platform.
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The structure of the SAM/SAH-binding Riboswitch.
Nucleic Acids Research, 2019Co-Authors: A.k. Weickhmann, Heiko Keller, Jan‐philip Wurm, Elisabeth Strebitzer, Juen, Johannes Kremser, Zasha Weinberg, Christoph Kreutz, Elke Duchardt-ferner, Jens WöhnertAbstract:S-adenosylmethionine (SAM) is a central metabolite since it is used as a methyl group donor in many different biochemical reactions. Many bacteria control intracellular SAM concentrations using Riboswitch-based mechanisms. A number of structurally different Riboswitch families specifically bind to SAM and mainly regulate the transcription or the translation of SAM-biosynthetic enzymes. In addition, a highly specific Riboswitch class recognizes S-adenosylhomocysteine (SAH)-the product of SAM-dependent methyl group transfer reactions-and regulates enzymes responsible for SAH hydrolysis. High-resolution structures are available for many of these Riboswitch classes and illustrate how they discriminate between the two structurally similar ligands SAM and SAH. The so-called SAM/SAH Riboswitch class binds both ligands with similar affinities and is structurally not yet characterized. Here, we present a high-resolution nuclear magnetic resonance structure of a member of the SAM/SAH-Riboswitch class in complex with SAH. Ligand binding induces pseudoknot formation and sequestration of the ribosome binding site. Thus, the SAM/SAH-Riboswitches are translational 'OFF'-switches. Our results establish a structural basis for the unusual bispecificity of this Riboswitch class. In conjunction with genomic data our structure suggests that the SAM/SAH-Riboswitches might be an evolutionary late invention and not a remnant of a primordial RNA-world as suggested for other Riboswitches.
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SAM-VI RNAs selectively bind S-adenosylmethionine and exhibit similarities to SAM-III Riboswitches
RNA Biology, 2018Co-Authors: Gayan Mirihana Arachchilage, Zasha Weinberg, Madeline E. Sherlock, Ronald R BreakerAbstract:ABSTRACTFive distinct Riboswitch classes that regulate gene expression in response to the cofactor S-adenosylmethionine (SAM) or its metabolic breakdown product S-adenosylhomocysteine (SAH) have been reported previously. Collectively, these SAM- or SAH-sensing RNAs constitute the most abundant collection of Riboswitches, and are found in nearly every major bacterial lineage. Here, we report a potential sixth member of this pervasive Riboswitch family, called SAM-VI, which is predominantly found in Bifidobacterium species. SAM-VI aptamers selectively bind the cofactor SAM and strongly discriminate against SAH. The consensus sequence and structural model for SAM-VI share some features with the consensus model for the SAM-III Riboswitch class, whose members are mainly found in lactic acid bacteria. However, there are sufficient differences between the two classes such that current bioinformatics methods separately cluster representatives of the two motifs. These findings highlight the abundance of RNA struct...
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bioinformatic analysis of Riboswitch structures uncovers variant classes with altered ligand specificity
Proceedings of the National Academy of Sciences of the United States of America, 2017Co-Authors: Zasha Weinberg, Madeline E. Sherlock, Ronald R Breaker, Christina E. Lünse, James W NelsonAbstract:Riboswitches are RNAs that form complex, folded structures that selectively bind small molecules or ions. As with certain groups of protein enzymes and receptors, some Riboswitch classes have evolved to change their ligand specificity. We developed a procedure to systematically analyze known Riboswitch classes to find additional variants that have altered their ligand specificity. This approach uses multiple-sequence alignments, atomic-resolution structural information, and Riboswitch gene associations. Among the discoveries are unique variants of the guanine Riboswitch class that most tightly bind the nucleoside 2′-deoxyguanosine. In addition, we identified variants of the glycine Riboswitch class that no longer recognize this amino acid, additional members of a rare flavin mononucleotide (FMN) variant class, and also variants of c-di-GMP-I and -II Riboswitches that might recognize different bacterial signaling molecules. These findings further reveal the diverse molecular sensing capabilities of RNA, which highlights the potential for discovering a large number of additional natural Riboswitch classes.
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Structural, Functional, and Taxonomic Diversity of Three PreQ1 Riboswitch Classes
Chemistry & Biology, 2014Co-Authors: Phillip J. Mccown, Zasha Weinberg, Jonathan J. Liang, Ronald R BreakerAbstract:Previously, two Riboswitch classes have been identified that sense and respond to the hypermodified nucleobase called prequeuosine1 (preQ1). The enormous expansion of available genomic DNA sequence data creates new opportunities to identify additional representatives of the known Riboswitch classes and to discover novel classes. We conducted bioinformatics searches on microbial genomic DNA data sets to discover numerous additional examples belonging to the two previously known Riboswitch classes for preQ1 (classes preQ1-I and preQ1-II), including some structural variants that further restrict ligand specificity. Additionally, we discovered a third preQ1-binding Riboswitch class (preQ1-III) that is structurally distinct from previously known classes. These findings demonstrate that numerous organisms monitor the concentrations of this modified nucleobase by exploiting one or more Riboswitch classes for this widespread compound.
Daniel A Lafontaine - One of the best experts on this subject based on the ideXlab platform.
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Probing of Nascent Riboswitch Transcripts.
Methods of Molecular Biology, 2020Co-Authors: Adrien Chauvier, Daniel A LafontaineAbstract:: The study of biologically significant and native structures is vital to characterize RNA-based regulatory mechanisms. Riboswitches are cis-acting RNA molecules that are involved in the biosynthesis and transport of cellular metabolites. Because Riboswitches regulate gene expression by modulating their structure, it is vital to employ native probing assays to determine how native Riboswitch structures perform highly efficient and specific ligand recognition. By employing RNase H probing, it is possible to determine the accessibility of specific RNA domains in various structural contexts. Herein, we describe how to employ RNase H probing to characterize nascent mRNA Riboswitch molecules as a way to obtain information regarding the Riboswitch regulation control mechanism.
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Single-Molecule Approaches for the Characterization of Riboswitch Folding Mechanisms.
Methods of Molecular Biology, 2020Co-Authors: Julien Boudreault, D. Cibran Perez-gonzalez, J. Carlos Penedo, Daniel A LafontaineAbstract:: Riboswitches are highly structured RNA molecules that control genetic expression by altering their structure as a function of metabolite binding. Accumulating evidence suggests that Riboswitch structures are highly dynamic and perform conformational exchange between structural states that are important for the outcome of genetic regulation. To understand how ligand binding influences the folding of Riboswitches, it is important to monitor in real time the Riboswitch folding pathway as a function of experimental conditions. Single-molecule FRET (sm-FRET) is unique among biophysical techniques to study Riboswitch conformational changes as it allows to both monitor steady-state populations of Riboswitch conformers and associated interconversion dynamics. Since FRET fluorophores can be attached to virtually any nucleotide position, FRET assays can be adapted to monitor specific conformational changes, thus enabling to deduce complex Riboswitch folding pathways. Herein, we show how to employ sm-FRET to study the folding pathway of the S-adenosylmethionine (SAM) and how this can be used to understand very specific conformational changes that are at the heart of Riboswitch regulation mechanism.
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Role of lysine binding residues in the global folding of the lysC Riboswitch.
RNA Biology, 2015Co-Authors: Erich Smith-peter, Anne-marie Lamontagne, Daniel A LafontaineAbstract:Riboswitches regulate gene expression by rearranging their structure upon metabolite binding. The lysine-sensing lysC Riboswitch is a rare example of an RNA aptamer organized around a 5-way helical junction in which ligand binding is performed exclusively through nucleotides located at the junction core. We have probed whether the nucleotides involved in ligand binding play any role in the global folding of the Riboswitch. As predicted, our findings indicate that ligand-binding residues are critical for the lysine-dependent gene regulation mechanism. We also find that these residues are not important for the establishment of key magnesium-dependent tertiary interactions, suggesting that folding and ligand recognition are uncoupled in this Riboswitch for the formation of specific interactions. However, FRET assays show that lysine binding results in an additional conformational change, indicating that lysine binding may also participate in a specific folding transition. Thus, in contrast to helical junctions being primary determinants in ribozymes and rRNA folding, we speculate that the helical junction of the lysine-sensing lysC Riboswitch is not employed as structural a scaffold to direct global folding, but rather has a different role in establishing RNA-ligand interactions required for Riboswitch regulation. Our work suggests that helical junctions may adopt different functions such as the coordination of global architecture or the formation of specific ligand binding site.
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A kissing loop is important for btuB Riboswitch ligand sensing and regulatory control.
Journal of Biological Chemistry, 2015Co-Authors: Antony Lussier, Laurene Bastet, Adrien Chauvier, Daniel A LafontaineAbstract:: RNA-based genetic regulation is exemplified by metabolite-binding Riboswitches that modulate gene expression through conformational changes. Crystal structures show that the Escherichia coli btuB Riboswitch contains a kissing loop interaction that is in close proximity to the bound ligand. To analyze the role of the kissing loop interaction in the Riboswitch regulatory mechanism, we used RNase H cleavage assays to probe the structure of nascent Riboswitch transcripts produced by the E. coli RNA polymerase. By monitoring the folding of the aptamer, kissing loop, and Riboswitch expression platform, we established the conformation of each structural component in the absence or presence of bound adenosylcobalamin. We found that the kissing loop interaction is not essential for ligand binding. However, we showed that kissing loop formation improves ligand binding efficiency and is required to couple ligand binding to the Riboswitch conformational changes involved in regulating gene expression. These results support a mechanism by which the btuB Riboswitch modulates the formation of a tertiary structure to perform metabolite sensing and regulate gene expression.
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dual acting Riboswitch control of translation initiation and mrna decay
Proceedings of the National Academy of Sciences of the United States of America, 2012Co-Authors: Mariepier Caron, Laurene Bastet, Antony Lussier, Maxime Simoneauroy, Eric Masse, Daniel A LafontaineAbstract:Riboswitches are mRNA regulatory elements that control gene expression by altering their structure in response to specific metabolite binding. In bacteria, Riboswitches consist of an aptamer that performs ligand recognition and an expression platform that regulates either transcription termination or translation initiation. Here, we describe a dual-acting Riboswitch from Escherichia coli that, in addition to modulating translation initiation, also is directly involved in the control of initial mRNA decay. Upon lysine binding, the lysC Riboswitch adopts a conformation that not only inhibits translation initiation but also exposes RNase E cleavage sites located in the Riboswitch expression platform. However, in the absence of lysine, the Riboswitch folds into an alternative conformation that simultaneously allows translation initiation and sequesters RNase E cleavage sites. Both regulatory activities can be individually inhibited, indicating that translation initiation and mRNA decay can be modulated independently using the same conformational switch. Because RNase E cleavage sites are located in the Riboswitch sequence, this Riboswitch provides a unique means for the Riboswitch to modulate RNase E cleavage activity directly as a function of lysine. This dual inhibition is in contrast to other Riboswitches, such as the thiamin pyrophosphate-sensing thiM Riboswitch, which triggers mRNA decay only as a consequence of translation inhibition. The Riboswitch control of RNase E cleavage activity is an example of a mechanism by which metabolite sensing is used to regulate gene expression of single genes or even large polycistronic mRNAs as a function of environmental changes.
Nils G Walter - One of the best experts on this subject based on the ideXlab platform.
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ligand modulates cross coupling between Riboswitch folding and transcriptional pausing
Molecular Cell, 2018Co-Authors: Julia R Widom, Charles L Brooks, Irina Artsimovitch, Yuri Nedialkov, Ryan L Hayes, Nils G WalterAbstract:Summary Numerous classes of Riboswitches have been found to regulate bacterial gene expression in response to physiological cues, offering new paths to antibacterial drugs. As common studies of isolated Riboswitches lack the functional context of the transcription machinery, we here combine single-molecule, biochemical, and simulation approaches to investigate the coupling between co-transcriptional folding of the pseudoknot-structured preQ1 Riboswitch and RNA polymerase (RNAP) pausing. We show that pausing at a site immediately downstream of the Riboswitch requires a ligand-free pseudoknot in the nascent RNA, a precisely spaced sequence resembling the pause consensus, and electrostatic and steric interactions with the RNAP exit channel. While interactions with RNAP stabilize the native fold of the Riboswitch, binding of the ligand signals RNAP release from the pause. Our results demonstrate that the nascent Riboswitch and its ligand actively modulate the function of RNAP and vice versa, a paradigm likely to apply to other cellular RNA transcripts.
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hierarchical mechanism of amino acid sensing by the t box Riboswitch
Nature Communications, 2018Co-Authors: Krishna C Suddala, Javier Cabellovillegas, Malgorzata Michnicka, Collin Marshall, Edward P Nikonowicz, Nils G WalterAbstract:In Gram-positive bacteria, T-box Riboswitches control gene expression to maintain the cellular pools of aminoacylated tRNAs essential for protein biosynthesis. Co-transcriptional binding of an uncharged tRNA to the Riboswitch stabilizes an antiterminator, allowing transcription read-through, whereas an aminoacylated tRNA does not. Recent structural studies have resolved two contact points between tRNA and Stem-I in the 5′ half of the T-box Riboswitch, but little is known about the mechanism empowering transcriptional control by a small, distal aminoacyl modification. Using single-molecule fluorescence microscopy, we have probed the kinetic and structural underpinnings of tRNA binding to a glycyl T-box Riboswitch. We observe a two-step mechanism where fast, dynamic recruitment of tRNA by Stem-I is followed by ultra-stable anchoring by the downstream antiterminator, but only without aminoacylation. Our results support a hierarchical sensing mechanism wherein dynamic global binding of the tRNA body is followed by localized readout of its aminoacylation status by snap-lock-based trapping. Riboswitches on 5′ ends of mRNAs are important for bacterial gene regulation. Here the authors probe the mechanism of a tRNA aminoacylation sensing T-box Riboswitch using single-molecule fluorescence microscopy to characterize dynamic solution conformations and heterogeneous tRNA binding kinetics.
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cooperative and directional folding of the preq1 Riboswitch aptamer domain
Journal of the American Chemical Society, 2011Co-Authors: Jun Feng, Nils G Walter, Charles L BrooksAbstract:Riboswitches are cis-acting RNA fragments that regulate gene expression by sensing cellular levels of the associated small metabolites. In bacteria, the class I preQ1 Riboswitch allows the fine-tuning of queuosine biosynthesis in response to the intracellular concentration of the queuosine anabolic intermediate preQ1. When binding preQ1, the aptamer domain undergoes a significant degree of secondary and tertiary structural rearrangement and folds into an H-type pseudoknot. Conformational “switching” of the Riboswitch aptamer domain upon recognizing its cognate metabolite plays a key role in the regulatory mechanism of the preQ1 Riboswitch. We investigate the folding mechanism of the preQ1 Riboswitch aptamer domain using all-atom Go-model simulations. The folding pathway of such a single domain is found to be cooperative and sequentially coordinated, as the folding proceeds in the 5′ → 3′ direction. This kinetically efficient folding mechanism suggests a fast ligand-binding response in competition with RN...
