Dual-specificity RNA aptamers enable manipulation of target-specific O-GlcNAcylation and unveil functions of O-GlcNAc on ß-catenin.

O-GlcNAc is a dynamic post-translational modification (PTM) that regulates protein functions. In studying the regulatory roles of O-GlcNAc, a major roadblock is the inability to change O-GlcNAcylation on a single protein at a time. Herein, we developed a dual RNA-aptamer-based approach that simultaneously targeted O-GlcNAc transferase (OGT) and ß-catenin, the key transcription factor of the Wnt signaling pathway, to selectively increase O-GlcNAcylation of the latter without affecting other OGT substrates. Using the OGT/ß-catenin dual-specificity aptamers, we found that O-GlcNAcylation of ß-catenin stabilizes the protein by inhibiting its interaction with ß-TrCP. O-GlcNAc also increases ß-catenin's interaction with EZH2, recruits EZH2 to promoters, and dramatically alters the transcriptome. Further, by coupling riboswitches or an inducible expression system to aptamers, we enabled inducible regulation of protein-specific O-GlcNAcylation. Together, our findings demonstrate the efficacy and versatility of dual-specificity aptamers for regulating O-GlcNAcylation on individual proteins.

Engineering and In Vivo Applications of Riboswitches.

Riboswitches are common gene regulatory units mostly found in bacteria that are capable of altering gene expression in response to a small molecule. These structured RNA elements consist of two modular subunits: an aptamer domain that binds with high specificity and affinity to a target ligand and an expression platform that transduces ligand binding to a gene expression output. Significant progress has been made in engineering novel aptamer domains for new small molecule inducers of gene expression. Modified expression platforms have also been optimized to function when fused with both natural and synthetic aptamer domains. As this field expands, the use of these privileged scaffolds has permitted the development of tools such as RNA-based fluorescent biosensors. In this review, we summarize the methods that have been developed to engineer new riboswitches and highlight applications of natural and synthetic riboswitches in enzyme and strain engineering, in controlling gene expression and cellular physiology, and in real-time imaging of cellular metabolites and signals.

Molecular dynamics in multidimensional space explains how mutations affect the association path of neomycin to a riboswitch.

Riboswitches are messenger RNA (mRNA) fragments binding specific small molecules to regulate gene expression. A synthetic N1 riboswitch, inserted into yeast mRNA controls the translation of a reporter gene in response to neomycin. However, its regulatory activity is sensitive to single-point RNA mutations, even those distant from the neomycin binding site. While the association paths of neomycin to N1 and its variants remain unknown, recent fluorescence kinetic experiments indicate a two-step process driven by conformational selection. This raises the question of which step is affected by mutations. To address this, we performed all-atom two-dimensional replica-exchange molecular dynamics simulations for N1 and U14C, U14C[Formula: see text], U15A, and A17G mutants, ensuring extensive conformational sampling of both RNA and neomycin. The obtained neomycin association and binding paths, along with multidimensional free-energy profiles, revealed a two-step binding mechanism, consisting of conformational selection and induced fit. Neomycin binds to a preformed N1 conformation upon identifying a stable upper stem and U-turn motif in the riboswitch hairpin. However, the positioning of neomycin in the binding site occurs at different RNA-neomycin distances for each mutant, which may explain their different regulatory activities. The subsequent induced fit arises from the interactions of the neomycin's N3 amino group with RNA, causing the G9 backbone to rearrange. In the A17G mutant, the critical C6-A17/G17 stacking forms at a closer RNA-neomycin distance compared to N1. These findings together with estimated binding free energies coincide with experiments and elucidate why the A17G mutation decreases and U15A enhances N1 activity in response to neomycin.

Direct and indirect control of Rho-dependent transcription termination by the Escherichia coli lysC riboswitch.

Bacterial riboswitches are molecular structures that play a crucial role in controlling gene expression to maintain cellular balance. The Escherichia coli lysC riboswitch has been previously shown to regulate gene expression through translation initiation and mRNA decay. Recent research suggests that lysC gene expression is also influenced by Rho-dependent transcription termination. Through a series of in silico, in vitro, and in vivo experiments, we provide experimental evidence that the lysC riboswitch directly and indirectly modulates Rho transcription termination. Our study demonstrates that Rho-dependent transcription termination plays a significant role in the cotranscriptional regulation of lysC expression. Together with previous studies, our work suggests that lysC expression is governed by a lysine-sensing riboswitch that regulates translation initiation, transcription termination, and mRNA degradation. Notably, both Rho and RNase E target the same region of the RNA molecule, implying that RNase E may degrade Rho-terminated transcripts, providing a means to selectively eliminate these incomplete messenger RNAs. Overall, this study sheds light on the complex regulatory mechanisms used by bacterial riboswitches, emphasizing the role of transcription termination in the control of gene expression and mRNA stability.

The Escherichia coli ribB riboswitch senses flavin mononucleotide within a defined transcriptional window.

Riboswitches are metabolite-binding RNA regulators that modulate gene expression at the levels of transcription and translation. One of the hallmarks of riboswitch regulation is that they undergo structural changes upon metabolite binding. While a lot of effort has been put to characterize how the metabolite is recognized by the riboswitch, there is still relatively little information regarding how ligand sensing is performed within a transcriptional context. Here, we study the ligand-dependent cotranscriptional folding of the FMN-sensing ribB riboswitch of Escherichia coli. Using RNase H assays to study nascent ribB riboswitch transcripts, DNA probes targeting the P1 and sequestering stems indicate that FMN binding leads to the protection of these regions from RNase H cleavage, consistent with the riboswitch inhibiting translation initiation when bound to FMN. Our results show that ligand sensing is strongly affected by the position of elongating RNA polymerase, which is defining an FMN binding transcriptional window that is bordered in its 3' extremity by a transcriptional pause site. Also, using successively overlapping DNA probes targeting a subdomain of the riboswitch, our data suggest the presence of a previously unsuspected helical region involving the 3' strand of the P1 stem. Our results show that this helical region is conserved across bacterial species, thus suggesting that this predicted structure, the anti*-P1 stem, is involved in the FMN-free conformation of the ribB riboswitch. Overall, our study further demonstrates that intricate folding strategies may be used by riboswitches to perform metabolite sensing during the transcriptional process.

Magnesium ions mitigate metastable states in the regulatory landscape of mRNA elements.

Residing in the 5' untranslated region of the mRNA, the 2'-deoxyguanosine (2'-dG) riboswitch mRNA element adopts an alternative structure upon binding of the 2'-dG molecule, which terminates transcription. RNA conformations are generally strongly affected by positively charged metal ions (especially Mg2+). We have quantitatively explored the combined effect of ligand (2'-dG) and Mg2+ binding on the energy landscape of the aptamer domain of the 2'-dG riboswitch with both explicit solvent all-atom molecular dynamics simulations (99 µsec aggregate sampling for the study) and selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE) experiments. We show that both ligand and Mg2+ are required for the stabilization of the aptamer domain; however, the two factors act with different modalities. The addition of Mg2+ remodels the energy landscape and reduces its frustration by the formation of additional contacts. In contrast, the binding of 2'-dG eliminates the metastable states by nucleating a compact core for the aptamer domain. Mg2+ ions and ligand binding are required to stabilize the least stable helix, P1 (which needs to unfold to activate the transcription platform), and the riboswitch core formed by the backbone of the P2 and P3 helices. Mg2+ and ligand also facilitate a more compact structure in the three-way junction region.

Structural idiosyncrasies of glycyl T-box riboswitches among pathogenic bacteria.

T-box riboswitches are widespread bacterial regulatory noncoding RNAs that directly interact with tRNAs and switch conformations to regulate the transcription or translation of genes related to amino acid metabolism. Recent studies in Bacilli have revealed the core mechanisms of T-boxes that enable multivalent, specific recognition of both the identity and aminoacylation status of the tRNA substrates. However, in-depth knowledge on a vast number of T-boxes in other bacterial species remains scarce, although a remarkable structural diversity, particularly among pathogens, is apparent. In the present study, analysis of T-boxes that control the transcription of glycyl-tRNA synthetases from four prominent human pathogens revealed significant structural idiosyncrasies. Nonetheless, these diverse T-boxes maintain functional T-box:tRNAGly interactions both in vitro and in vivo. Probing analysis not only validated recent structural observations, but also expanded our knowledge on the substantial diversities among T-boxes and suggest interesting distinctions from the canonical Bacilli T-boxes. Surprisingly, some glycyl T-boxes seem to redirect the T-box trajectory in the absence of recognizable K-turns or contain Stem II modules that are generally absent in glycyl T-boxes. These results consolidate the notion of a lineage-specific diversification and elaboration of the T-box mechanism and corroborate the potential of T-boxes as promising species-specific RNA targets for next-generation antibacterial compounds.

Cellular Adaptations to Cytoplasmic Mg2+ Limitation.

Mg2+ is the most abundant divalent cation in living cells. It is essential for charge neutralization, macromolecule stabilization, and the assembly and activity of ribosomes and as a cofactor for enzymatic reactions. When experiencing low cytoplasmic Mg2+, bacteria adopt two main strategies: They increase the abundance and activity of Mg2+ importers and decrease the abundance of Mg2+-chelating ATP and rRNA. These changes reduce regulated proteolysis by ATP-dependent proteases and protein synthesis in a systemic fashion. In many bacterial species, the transcriptional regulator PhoP controls expression of proteins mediating these changes. The 5' leader region of some mRNAs responds to low cytoplasmic Mg2+ or to disruptions in translation of open reading frames in the leader regions by furthering expression of the associated coding regions, which specify proteins mediating survival when the cytoplasmic Mg2+ concentration is low. Microbial species often utilize similar adaptation strategies to cope with low cytoplasmic Mg2+ despite relying on different genes to do so.

Ligand Modulates Cross-Coupling between Riboswitch Folding and Transcriptional Pausing.

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.

Flipping the script: Understanding riboswitches from an alternative perspective.

Riboswitches are broadly distributed regulatory elements most frequently found in the 5'-leader sequence of bacterial mRNAs that regulate gene expression in response to the binding of a small molecule effector. The occupancy status of the ligand-binding aptamer domain manipulates downstream information in the message that instructs the expression machinery. Currently, there are over 55 validated riboswitch classes, where each class is defined based on the identity of the ligand it binds and/or sequence and structure conservation patterns within the aptamer domain. This classification reflects an "aptamer-centric" perspective that dominates our understanding of riboswitches. In this review, we propose a conceptual framework that groups riboswitches based on the mechanism by which RNA manipulates information directly instructing the expression machinery. This scheme does not replace the established aptamer domain-based classification of riboswitches but rather serves to facilitate hypothesis-driven investigation of riboswitch regulatory mechanisms. Based on current bioinformatic, structural, and biochemical studies of a broad spectrum of riboswitches, we propose three major mechanistic groups: (1) "direct occlusion", (2) "interdomain docking", and (3) "strand exchange". We discuss the defining features of each group, present representative examples of riboswitches from each group, and illustrate how these RNAs couple small molecule binding to gene regulation. While mechanistic studies of the occlusion and docking groups have yielded compelling models for how these riboswitches function, much less is known about strand exchange processes. To conclude, we outline the limitations of our mechanism-based conceptual framework and discuss how critical information within riboswitch expression platforms can inform gene regulation.

Probing RNA structure and dynamics using nanopore and next generation sequencing.

It has become increasingly evident that the structures RNAs adopt are conformationally dynamic; the various structured states that RNAs sample govern their interactions with other nucleic acids, proteins, and ligands to regulate a myriad of biological processes. Although several biophysical approaches have been developed and used to study the dynamic landscape of structured RNAs, technical limitations have limited their application to all classes of RNA due to variable size and flexibility. Recent advances combining chemical probing experiments with next-generation- and direct sequencing have emerged as an alternative approach to exploring the conformational dynamics of RNA. In this review, we provide a methodological overview of the sequencing-based techniques used to study RNA conformational dynamics. We discuss how different techniques have enabled us to better understand the propensity of RNAs from a variety of different classes to sample multiple conformational states. Finally, we present examples of the ways these techniques have reshaped how we think about RNA structure.

Translation regulation by a guanidine-II riboswitch is highly tunable in sensitivity, dynamic range, and apparent cooperativity.

Riboswitches function as important translational regulators in bacteria. Comprehensive mutational analysis of transcriptional riboswitches has been used to probe the energetic intricacies of interplay between the aptamer and expression platform, but translational riboswitches have been inaccessible to massively parallel techniques. The guanidine-II (gdm-II) riboswitch is an exclusively translational class. We have integrated RelE cleavage with next-generation sequencing to quantify ligand-dependent changes in translation initiation for all single and double mutations of the Pseudomonas aeruginosa gdm-II riboswitch, a total of more than 23,000 variants. This extensive mutational analysis is consistent with the prominent features of the bioinformatic consensus. These data indicate, unexpectedly, that direct sequestration of the Shine-Dalgarno sequence is dispensable for riboswitch function. Additionally, this comprehensive data set reveals important positions not identified in previous computational and crystallographic studies. Mutations in the variable linker region stabilize alternate conformations. The double mutant data reveal the functional importance of the previously modeled P0b helix formed by the 5' and 3' tails that serves as the basis for translational control. Additional mutations to GU wobble base pairs in both P1 and P2 reveal how the apparent cooperativity of the system involves an intricate network of communication between the two binding sites. This comprehensive examination of a translational riboswitch's expression platform illuminates how the riboswitch is precisely tuned and tunable with regard to ligand sensitivity, the amplitude of expression between ON and OFF states, and the cooperativity of ligand binding.

Rational design of eukaryotic riboswitches that up-regulate IRES-mediated translation initiation with high switching efficiency through a kinetic trapping mechanism in vitro.

In general, riboswitches functioning through a cotranscriptional kinetic trapping mechanism (kt-riboswitches) show higher switching efficiencies in response to practical concentrations of their ligand molecules than eq-riboswitches, which function by an equilibrium mechanism. However, the former have been much more difficult to design due to their more complex mechanism. We here successfully developed a rational strategy for constructing eukaryotic kt-riboswitches that ligand-dependently enhance translation initiation mediated by an internal ribosome entry site (IRES). This was achieved both by utilizing some predicted structural features of a highly efficient bacterial kt-riboswitch identified through screening and by completely decoupling an aptamer domain from the IRES. Three kt-riboswitches optimized through this strategy, each responding to a different ligand, exhibited three- to sevenfold higher induction ratios (up to ∼90) than previously optimized eq-riboswitches regulating the same IRES-mediated translation in wheat germ extract. Because the IRES used functions well in various eukaryotic expression systems, these types of kt-riboswitches are expected to serve as major eukaryotic gene regulators based on RNA. In addition, the present strategy could be applied to the rational construction of other types of kt-riboswitches, including those functioning in bacterial expression systems.

Structure and ion-dependent folding of k-junctions.

k-Junctions are elaborated forms of kink turns with an additional helix on the nonbulged strand, thus forming a three-way helical junction. Two were originally identified in the structures of Arabidopsis and Escherichia coli thiamine pyrophosphate (TPP) riboswitches, and another called DUF-3268 was tentatively identified from sequence information. In this work we show that the Arabidopsis and E. coli riboswitch k-junctions fold in response to the addition of magnesium or sodium ions, and that atomic mutations that should disrupt key hydrogen bonding interactions greatly impair folding. Using X-ray crystallography, we have determined the structure of the DUF-3268 RNA and thus confirmed that it is a k-junction. It also folds upon the addition of metal ions, though requiring a 40-fold lower concentration of either divalent or monovalent ions. The key difference between the DUF-3268 and riboswitch k-junctions is the lack of nucleotides inserted between G1b and A2b in the former. We show that this insertion is primarily responsible for the difference in folding properties. Finally, we show that the DUF-3268 can functionally substitute for the k-junction in the E. coli TPP riboswitch such that the chimera can bind the TPP ligand, although less avidly.

A computational approach for the identification of distant homologs of bacterial riboswitches based on inverse RNA folding.

Riboswitches are conserved structural ribonucleic acid (RNA) sensors that are mainly found to regulate a large number of genes/operons in bacteria. Presently, >50 bacterial riboswitch classes have been discovered, but only the thiamine pyrophosphate riboswitch class is detected in a few eukaryotes like fungi, plants and algae. One of the most important challenges in riboswitch research is to discover existing riboswitch classes in eukaryotes and to understand the evolution of bacterial riboswitches. However, traditional search methods for riboswitch detection have failed to detect eukaryotic riboswitches besides just one class and any distant structural homologs of riboswitches. We developed a novel approach based on inverse RNA folding that attempts to find sequences that match the shape of the target structure with minimal sequence conservation based on key nucleotides that interact directly with the ligand. Then, to support our matched candidates, we expanded the results into a covariance model representing similar sequences preserving the structure. Our method transforms a structure-based search into a sequence-based search that considers the conservation of secondary structure shape and ligand-binding residues. This method enables us to identify a potential structural candidate in fungi that could be the distant homolog of bacterial purine riboswitches. Further, phylogenomic analysis and evolutionary distribution of this structural candidate indicate that the most likely point of origin of this structural candidate in these organisms is associated with the loss of traditional purine riboswitches. The computational approach could be applicable to other domains and problems in RNA research.

Metabolism of Free Guanidine in Bacteria Is Regulated by a Widespread Riboswitch Class.

The guanidyl moiety is a component of fundamental metabolites, including the amino acid arginine, the energy carrier creatine, and the nucleobase guanine. Curiously, reports regarding the importance of free guanidine in biology are sparse, and no biological receptors that specifically recognize this compound have been previously identified. We report that many members of the ykkC motif RNA, the longest unresolved riboswitch candidate, naturally sense and respond to guanidine. This RNA is found throughout much of the bacterial domain of life, where it commonly controls the expression of proteins annotated as urea carboxylases and multidrug efflux pumps. Our analyses reveal that these proteins likely function as guanidine carboxylases and guanidine transporters, respectively. Furthermore, we demonstrate that bacteria are capable of endogenously producing guanidine. These and related findings demonstrate that free guanidine is a biologically relevant compound, and several gene families that can alleviate guanidine toxicity exist.

Structure and function analysis of a type III preQ1-I riboswitch from Escherichia coli reveals direct metabolite sensing by the Shine-Dalgarno sequence.

Riboswitches are small noncoding RNAs found primarily in the 5' leader regions of bacterial messenger RNAs where they regulate expression of downstream genes in response to binding one or more cellular metabolites. Such noncoding RNAs are often regulated at the translation level, which is thought to be mediated by the accessibility of the Shine-Dalgarno sequence (SDS) ribosome-binding site. Three classes (I-III) of prequeuosine1 (preQ1)-sensing riboswitches are known that control translation. Class I is divided into three subtypes (types I-III) that have diverse mechanisms of sensing preQ1, which is involved in queuosine biosynthesis. To provide insight into translation control, we determined a 2.30 Å-resolution cocrystal structure of a class I type III preQ1-sensing riboswitch identified in Escherichia coli (Eco) by bioinformatic searches. The Eco riboswitch structure differs from previous preQ1 riboswitch structures because it has the smallest naturally occurring aptamer and the SDS directly contacts the preQ1 metabolite. We validated structural observations using surface plasmon resonance and in vivo gene-expression assays, which showed strong switching in live E. coli. Our results demonstrate that the Eco riboswitch is relatively sensitive to mutations that disrupt noncanonical interactions that form the pseudoknot. In contrast to type II preQ1 riboswitches, a kinetic analysis showed that the type III Eco riboswitch strongly prefers preQ1 over the chemically similar metabolic precursor preQ0. Our results reveal the importance of noncanonical interactions in riboswitch-driven gene regulation and the versatility of the class I preQ1 riboswitch pseudoknot as a metabolite-sensing platform that supports SDS sequestration.

The current riboswitch landscape in Clostridioides difficile.

Riboswitches are 5' RNA regulatory elements that are capable of binding to various ligands, such as small metabolites, ions and tRNAs, leading to conformational changes and affecting gene transcription or translation. They are widespread in bacteria and frequently control genes that are essential for the survival or virulence of major pathogens. As a result, they represent promising targets for the development of new antimicrobial treatments. Clostridioides difficile, a leading cause of antibiotic-associated nosocomial diarrhoea in adults, possesses numerous riboswitches in its genome. Accumulating knowledge of riboswitch-based regulatory mechanisms provides insights into the potential therapeutic targets for treating C. difficile infections. This review offers an in-depth examination of the current state of knowledge regarding riboswitch-mediated regulation in C. difficile, highlighting their importance in bacterial adaptability and pathogenicity. Particular attention is given to the ligand specificity and function of known riboswitches in this bacterium. The review also discusses the recent progress that has been made in the development of riboswitch-targeting compounds as potential treatments for C. difficile infections. Future research directions are proposed, emphasizing the need for detailed structural and functional analyses of riboswitches to fully harness their regulatory capabilities for developing new antimicrobial strategies.

Use of CRISPR interference for efficient and rapid gene inactivation in Fusobacterium nucleatum.

Gene inactivation by creating in-frame deletion mutations in Fusobacterium nucleatum is time consuming, and most fusobacterial strains are genetically intractable. Addressing these problems, we introduced a riboswitch-based inducible CRISPR interference (CRISPRi) system. This system employs the nuclease-inactive Streptococcus pyogenes Cas9 protein (dCas9), specifically guided to the gene of interest by a constantly expressed single-guide RNA (sgRNA). Mechanistically, this dCas9-sgRNA complex serves as an insurmountable roadblock for RNA polymerase, thus repressing the target gene transcription. Leveraging this system, we first examined two non-essential genes, ftsX and radD, which are pivotal for fusobacterial cytokinesis and coaggregation. Upon adding the inducer, theophylline, ftsX suppression caused filamentous cell formation akin to chromosomal ftsX deletion, while targeting radD significantly reduced RadD protein levels, abolishing RadD-mediated coaggregation. The system was then extended to probe essential genes bamA and ftsZ, which are vital for outer membrane biogenesis and cell division. Impressively, bamA suppression disrupted membrane integrity and bacterial separation, stalling growth, while ftsZ targeting yielded elongated cells in broth with compromised agar growth. Further studies on F. nucleatum clinical strain CTI-2 and Fusobacterium periodonticum revealed reduced indole synthesis when targeting tnaA. Moreover, silencing clpB in F. periodonticum decreased ClpB, increasing thermal sensitivity. In summary, our CRISPRi system streamlines gene inactivation across various fusobacterial strains.IMPORTANCEHow can we effectively investigate the gene functions in Fusobacterium nucleatum, given the dual challenges of gene inactivation and the inherent genetic resistance of many strains? Traditional methods have been cumbersome and often inadequate. Addressing this, our work introduces a novel inducible CRISPR interference (CRISPRi) system in which dCas9 expression is controlled at the translation level by a theophylline-responsive riboswitch unit, and single-guide RNA expression is driven by the robust, constitutive rpsJ promoter. This approach simplifies gene inactivation in the model organism (ATCC 23726) and extends its application to previously considered genetically intractable strains like CTI-2 and Fusobacterium periodonticum. With CRISPRi's potential, it is a pivotal tool for in-depth genetic studies into fusobacterial pathogenesis, potentially unlocking targeted therapeutic strategies.

Corrin Ring Modifications Reveal the Chemical and Spatial Requirements for the B12-btuB Riboswitch Interaction.

The btuB riboswitch is a regulatory RNA sequence controlling gene expression of the outer membrane B12 transport protein BtuB by specifically binding coenzyme B12 (AdoCbl) as its natural ligand. The B12 sensing riboswitch class is known to accept various B12 derivatives, leading to a division into two riboswitch subclasses, dependent on the size of the apical ligand. Here we focus on the role of side chains b and e on affinity and proper recognition, i. e. correct structural switch of the btuB RNA, which belongs to the AdoCbl-binding class I. Chemical modification of these side chains disturbs crucial hydrogen bonds and/or electrostatic interactions with the RNA, its effect on both affinity and switching being monitored by in-line probing. Chemical modifications at sidechain b of vitamin B12 show larger effects indicating crucial B12-RNA interactions. When introducing the same modification to AdoCbl the influence of any side-chain modification tested is reduced. This renders the impact of the adenosyl-ligand for B12-btuB riboswitch recognition clearly beyond the known role in affinity.