A structural intermediate pre-organizes the add adenine riboswitch for ligand recognition

Riboswitches are RNA sequences that regulate gene expression by undergoing structural changes upon the specific binding of cellular metabolites. Crystal structures of purine-sensing riboswitches have revealed an intricate network of interactions surrounding the ligand in the bound complex. The mechanistic details about how the aptamer folding pathway is involved in the formation of the metabolite binding site have been previously shown to be highly important for the riboswitch regulatory activity. Here, a combination of single-molecule FRET and SHAPE assays have been used to characterize the folding pathway of the adenine riboswitch from Vibrio vulnificus. Experimental evidences suggest a folding process characterized by the presence of a structural intermediate involved in ligand recognition. This intermediate state acts as an open conformation to ensure ligand accessibility to the aptamer and folds into a structure nearly identical to the ligand-bound complex through a series of structural changes. This study demonstrates that the add riboswitch relies on the folding of a structural intermediate that pre-organizes the aptamer global structure and the ligand binding site to allow efficient metabolite sensing and riboswitch genetic regulation.     

Folding of the SAM-I riboswitch: A tale with a twist

Riboswitches are ligand-dependent RNA genetic regulators that control gene expression by altering their structures. The elucidation of riboswitch conformational changes before and after ligand recognition is crucial to understand how riboswitches can achieve high ligand binding affinity and discrimination against cellular analogs. The detailed characterization of riboswitch folding pathways suggest that they may use their intrinsic conformational dynamics to sample a large array of structures, some of which being nearly identical to ligand-bound molecules. Some of these structural conformers can be "captured" upon ligand binding, which is crucial for the outcome of gene regulation. Recent studies about the SAM-I riboswitch have revealed unexpected and previously unknown RNA folding mechanisms. For instance, the observed helical twist of the P1 stem upon ligand binding to the SAM-I aptamer adds a new element in the repertoire of RNA strategies for recognition of small metabolites. From an RNA folding perspective, these findings also strongly indicate that the SAM-I riboswitch could achieve ligand recognition by using an optimized combination of conformational capture and induced-fit approaches, a feature that may be shared by other RNA regulatory sequences.     

Computational analysis of riboswitch-based regulation

Advances in computational analysis of riboswitches in the last decade have contributed greatly to our understanding of riboswitch regulatory roles and mechanisms. Riboswitches were originally discovered as part of the sequence analysis of the 5′-untranslated region of mRNAs in the hope of finding novel gene regulatory sites, and the existence of structural RNAs appeared to be a spurious phenomenon. As more riboswitches were discovered, they illustrated the diversity and adaptability of these RNA regulatory sequences. The fact that a chemically monotonous molecule like RNA can discern a wide range of substrates and exert a variety of regulatory mechanisms was subsequently demonstrated in diverse genomes and has hastened the development of sophisticated algorithms for their analysis and prediction. In this review, we focus on some of the computational tools for riboswitch detection and secondary structure prediction. The study of this simple yet efficient form of gene regulation promises to provide a more complete picture of a world that RNA once dominated and allows rational design of artificial riboswitches. This article is part of a Special Issue entitled: Riboswitches.     

Rational design of SAM analogues targeting SAM-II riboswitch aptamer

Riboswitches are noncoding RNA elements embedded in 5′-untranslated region of many bacterial mRNAs regulating gene expression in response to essential metabolites. They are unique from other RNA targets because they have evolved to form specific structural receptors for the purpose of binding small molecular metabolites suggesting that structure-based rational drug design approach may be used in designing metabolite mimics targeting riboswitches. We have developed a fluorescence binding assay for SAM-II riboswitch aptamer and identified an S-adenosylmethionine (SAM) analogue that selectively binds to SAM-II riboswitch aptamer with comparable binding affinity to its native metabolite using structure-based design approach.     

Riboswitches as versatile gene control elements

Riboswitches are structured elements typically found in the 5' untranslated regions of mRNAs, where they regulate gene expression by binding to small metabolites. In all examples studied to date, these RNA control elements do not require the involvement of protein factors for metabolite binding. Riboswitches appear to be pervasive in eubacteria, suggesting that this form of regulation is an important mechanism by which metabolic genes are controlled. Recently discovered riboswitch classes have surprisingly complex mechanisms for regulating gene expression and new high-resolution structural models of these RNAs provide insight into the molecular details of metabolite recognition by natural RNA aptamers.     

Themes and variations in riboswitch structure and function

The complexity of gene expression control by non-coding RNA has been highlighted by the recent progress in the field of riboswitches. Discovered a decade ago, riboswitches represent a diverse group of non-coding mRNA regions that possess a unique ability to directly sense cellular metabolites and modulate gene expression through formation of alternative metabolite-free and metabolite-bound conformations. Such protein-free metabolite sensing domains utilize sophisticated three-dimensional folding of RNA molecules to discriminate between a cognate ligand from related compounds so that only the right ligand would trigger a genetic response. Given the variety of riboswitch ligands ranging from small cations to large coenzymes, riboswitches adopt a great diversity of structures. Although many riboswitches share structural principles to build metabolite-competent folds, form precise ligand-binding pockets, and communicate a ligand-binding event to downstream regulatory regions, virtually all riboswitch classes possess unique features for ligand recognition, even those tuned to recognize the same metabolites. Here we present an overview of the biochemical and structural research on riboswitches with a major focus on common principles and individual characteristics adopted by these regulatory RNA elements during evolution to specifically target small molecules and exert genetic responses. This article is part of a Special Issue entitled: Riboswitches.     

The THI-box riboswitch, or how RNA binds thiamin pyrophosphate

Riboswitches are genetic control elements present mainly in the 5' untranslated regions of messenger RNAs that, upon binding of a small metabolite (like some vitamins, amino acids, and nucleobases), undergo conformational changes, affecting the expression of downstream genes. Structural studies of riboswitches are important for understanding how they recognize their ligands with high specificity and affinity. The thiamin pyrophosphate binding riboswitch (THI- box) is widely distributed in the three kingdoms of life and is involved in very distinct modes of gene regulation. Three recent THI-box structural analyses revealed how polyanionic RNA is able to bind a molecule with a negatively charged pyrophosphate group like thiamin pyrophosphate (TPP) and how it can discriminate between TPP and monophosphorylated analog molecules. These studies give insight into the genetic regulatory mechanisms in which the THI-box is involved.     

Riboswitches as antibacterial drug targets

New validated cellular targets are needed to reinvigorate antibacterial drug discovery. This need could potentially be filled by riboswitches-messenger RNA (mRNA) structures that regulate gene expression in bacteria. Riboswitches are unique among RNAs that serve as drug targets in that they have evolved to form structured and highly selective receptors for small drug-like metabolites. In most cases, metabolite binding to the receptor represses the expression of the gene(s) encoded by the mRNA. If a new metabolite analog were designed that binds to the receptor, the gene(s) regulated by that riboswitch could be repressed, with a potentially lethal effect to the bacteria. Recent work suggests that certain antibacterial compounds discovered decades ago function at least in part by targeting riboswitches. Herein we will summarize the experiments validating riboswitches as drug targets, describe the existing technology for riboswitch drug discovery and discuss the challenges that may face riboswitch drug discoverers.     

Idiosyncratically tuned switching behavior of riboswitch aptamer domains revealed by comparative small-angle X-ray scattering analysis

Riboswitches are structured mRNA elements that regulate gene expression upon binding specific cellular metabolites. It is thought that the highly conserved metabolite-binding domains of riboswitches undergo conformational change upon binding their cognate ligands. To investigate the generality of such a mechanism, we employed small-angle X-ray scattering (SAXS). We probed the nature of the global metabolite-induced response of the metabolite-binding domains of four different riboswitches that bind, respectively, thiamine pyrophosphate (TPP), flavin mononucleotide (FMN), lysine, and S-adenosyl methionine (SAM). We find that each RNA is unique in its global structural response to metabolite. Whereas some RNAs exhibit distinct free and bound conformations, others are globally insensitive to the presence of metabolite. Thus, a global conformational change of the metabolite-binding domain is not a requirement for riboswitch function. It is possible that the range of behaviors observed by SAXS, rather than being a biophysical idiosyncrasy, reflects adaptation of riboswitches to the regulatory requirements of their individual genomic context.