ykkC riboswitches employ an add-on helix to adjust specificity for polyanionic ligands.

The ykkC family of bacterial riboswitches combines several widespread classes that have similar secondary structures and consensus motifs but control different genes in response to different cellular metabolites. Here we report the crystal structures of two distinct ykkC riboswitches specifically bound to their cognate ligand ppGpp, a second messenger involved in stress response, or PRPP, a precursor in purine biosynthesis. Both RNAs adopt similar structures and contain a conserved core previously observed in the guanidine-specific ykkC riboswitch. However, ppGpp and PRPP riboswitches uniquely employ an additional helical element that joins the ends of the ligand-sensing domains and creates a tunnel for direct and Mg2+-mediated binding of ligands. Mutational and footprinting experiments highlight the importance of conserved nucleotides forming the tunnel and long-distance contacts for ligand binding and genetic response. Our work provides new insights into the specificity of riboswitches and gives a unique opportunity for future studies of RNA evolution.

RNA-Puzzles Round II: assessment of RNA structure prediction programs applied to three large RNA structures.

This paper is a report of a second round of RNA-Puzzles, a collective and blind experiment in three-dimensional (3D) RNA structure prediction. Three puzzles, Puzzles 5, 6, and 10, represented sequences of three large RNA structures with limited or no homology with previously solved RNA molecules. A lariat-capping ribozyme, as well as riboswitches complexed to adenosylcobalamin and tRNA, were predicted by seven groups using RNAComposer, ModeRNA/SimRNA, Vfold, Rosetta, DMD, MC-Fold, 3dRNA, and AMBER refinement. Some groups derived models using data from state-of-the-art chemical-mapping methods (SHAPE, DMS, CMCT, and mutate-and-map). The comparisons between the predictions and the three subsequently released crystallographic structures, solved at diffraction resolutions of 2.5-3.2 Å, were carried out automatically using various sets of quality indicators. The comparisons clearly demonstrate the state of present-day de novo prediction abilities as well as the limitations of these state-of-the-art methods. All of the best prediction models have similar topologies to the native structures, which suggests that computational methods for RNA structure prediction can already provide useful structural information for biological problems. However, the prediction accuracy for non-Watson-Crick interactions, key to proper folding of RNAs, is low and some predicted models had high Clash Scores. These two difficulties point to some of the continuing bottlenecks in RNA structure prediction. All submitted models are available for download at http://ahsoka.u-strasbg.fr/rnapuzzles/.

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.

Cooperativity and Allostery in RNA Systems.

Allostery is among the most basic biological principles employed by biological macromolecules to achieve a biologically active state in response to chemical cues. Although initially used to describe the impact of small molecules on the conformation and activity of protein enzymes, the definition of this term has been significantly broadened to describe long-range conformational change of macromolecules in response to small or large effectors. Such a broad definition could be applied to RNA molecules, which do not typically serve as protein-free cellular enzymes but fold and form macromolecular assemblies with the help of various ligand molecules, including ions and proteins. Ligand-induced allosteric changes in RNA molecules are often accompanied by cooperative interactions between RNA and its ligand, thus streamlining the folding and assembly pathways. This chapter provides an overview of the interplay between cooperativity and allostery in RNA systems and outlines methods to study these two biological principles.

Inhibitors of bacterial H2S biogenesis targeting antibiotic resistance and tolerance.

Emergent resistance to all clinical antibiotics calls for the next generation of therapeutics. Here we report an effective antimicrobial strategy targeting the bacterial hydrogen sulfide (H2S)-mediated defense system. We identified cystathionine γ-lyase (CSE) as the primary generator of H2S in two major human pathogens, Staphylococcus aureus and Pseudomonas aeruginosa, and discovered small molecules that inhibit bacterial CSE. These inhibitors potentiate bactericidal antibiotics against both pathogens in vitro and in mouse models of infection. CSE inhibitors also suppress bacterial tolerance, disrupting biofilm formation and substantially reducing the number of persister bacteria that survive antibiotic treatment. Our results establish bacterial H2S as a multifunctional defense factor and CSE as a drug target for versatile antibiotic enhancers.

Preparation and Crystallization of Riboswitches.

Recent studies have revealed that the majority of biological processes are controlled by noncoding RNAs. Among many classes of noncoding RNAs, metabolite-sensing segments of mRNAs called riboswitches are unique. Discovered over a decade ago in all three kingdoms of life, riboswitches specifically and directly interact with various metabolites and regulate expression of multiple genes, often associated with metabolism and transport of small molecules. Thus, riboswitches do not depend on proteins for binding to small molecules and play a role as both metabolite sensors and effectors of gene control. Riboswitches are typically located in the untranslated regions of mRNAs where they form alternative structures in the presence and absence of the ligand and modulate expression of genes through the formation of regulatory elements. To understand the mechanism of the riboswitch-driven gene control, it is important to elucidate how riboswitches interact with cognate and discriminate against non-cognate ligands. Here we outline the methodology to synthesize riboswitch RNAs and prepare riboswitch-ligand complexes for crystallographic and biochemical studies. The chapter describes how to design, prepare, and conduct crystallization screening of riboswitch-ligand complexes. The methodology was refined on crystallographic studies of several riboswitches and can be employed for other types of RNA molecules.

Cooperativity, allostery and synergism in ligand binding to riboswitches.

Recent progress in identification and characterization of novel types of non-coding RNAs has proven that RNAs carry out a variety of cellular functions ranging from scaffolding to gene expression control. In both prokaryotic and eukaryotic cells, several classes of non-coding RNAs control expression of dozens of genes in response to specific cues. One of the most interesting and outstanding questions in the RNA field is whether regulatory RNAs are capable of employing basic biological concepts, such as allostery and cooperativity, previously attributed to the function of proteins. Aside from regulatory RNAs that form complementary base pairing with their nucleic acid targets, several RNA classes modulate gene expression via molecular mechanisms which can be paralleled to protein-mediated regulation. Among these RNAs are riboswitches, metabolite-sensing non-coding regulatory elements that adopt intrinsic three-dimensional structures and specifically bind various small molecule ligands. These characteristics of riboswitches make them well-suited for complex regulatory responses observed in allosteric and cooperative protein systems. Here we present an overview of the biochemical, genetic, and structural studies of riboswitches with a major focus on complex regulatory mechanisms and biological principles utilized by riboswitches for such genetic modulation.

Structural and Dynamic Basis for Low-Affinity, High-Selectivity Binding of L-Glutamine by the Glutamine Riboswitch.

Naturally occurring L-glutamine riboswitches occur in cyanobacteria and marine metagenomes, where they reside upstream of genes involved in nitrogen metabolism. By combining X-ray, NMR, and MD, we characterized an L-glutamine-dependent conformational transition in the Synechococcus elongatus glutamine riboswitch from tuning fork to L-shaped alignment of stem segments. This transition generates an open ligand-binding pocket with L-glutamine selectivity enforced by Mg(2+)-mediated intermolecular interactions. The transition also stabilizes the P1 helix through a long-range "linchpin" Watson-Crick G-C pair-capping interaction, while melting a short helix below P1 potentially capable of modulating downstream readout. NMR data establish that the ligand-free glutamine riboswitch in Mg(2+) solution exists in a slow equilibrium between flexible tuning fork and a minor conformation, similar, but not identical, to the L-shaped bound conformation. We propose that an open ligand-binding pocket combined with a high conformational penalty for forming the ligand-bound state provide mechanisms for reducing binding affinity while retaining high selectivity.

Structure and function of pseudoknots involved in gene expression control.

Natural RNA molecules can have a high degree of structural complexity but even the most complexly folded RNAs are assembled from simple structural building blocks. Among the simplest RNA elements are double-stranded helices that participate in the formation of different folding topologies and constitute the major fraction of RNA structures. One common folding motif of RNA is a pseudoknot, defined as a bipartite helical structure formed by base-pairing of the apical loop in the stem-loop structure with an outside sequence. Pseudoknots constitute integral parts of the RNA structures essential for various cellular activities. Among many functions of pseudoknotted RNAs is feedback regulation of gene expression, carried out through specific recognition of various molecules. Pseudoknotted RNAs autoregulate ribosomal and phage protein genes in response to downstream encoded proteins, while many metabolic and transport genes are controlled by cellular metabolites interacting with pseudoknotted RNA elements from the riboswitch family. Modulation of some genes also depends on metabolite-induced messenger RNA (mRNA) cleavage performed by pseudoknotted ribozymes. Several regulatory pseudoknots have been characterized biochemically and structurally in great detail. These studies have demonstrated a plethora of pseudoknot-based folds and have begun uncovering diverse molecular principles of the ligand-dependent gene expression control. The pseudoknot-mediated mechanisms of gene control and many unexpected and interesting features of the regulatory pseudoknots have significantly advanced our understanding of the genetic circuits and laid the foundation for modulation of their outcomes.

Structural insights into ligand binding and gene expression control by an adenosylcobalamin riboswitch.

Coenzyme B(12) has a key role in various enzymatic reactions and controls expression of bacterial genes through riboswitches. Here we report the crystal structure of the Symbiobacterium thermophilum B(12) riboswitch bound to its ligand adenosylcobalamin. The riboswitch forms a unique junctional structure with a large ligand-binding pocket tailored for specific recognition of the adenosyl moiety and flanked by structural elements that stabilize the regulatory region and enable control of gene expression.