The pbuE adenine riboswitch undergoes metal ion-dependent folding that involves a loop-loop interaction. Binding of 2-aminopurine to the aptamer domain strongly correlates with the ability of the loops to interact, and single-molecule FRET studies reveal that folding proceeds via a discrete intermediate. Folding occurs in the absence of adenine ligand, but ligand binding stabilizes the folded structure by increasing the folding rate and decreasing the unfolding rate, and it lowers the magnesium ion concentration required to promote the loop-loop interaction. Individual aptamer molecules exhibit great heterogeneity in folding and unfolding rates, but this is reduced in the presence of adenine. In the full riboswitch, the adenine binding domain fails to fold because of conformational competition by the terminator stem. Thus, riboswitch function should depend on the relative rates of ligand binding and the transcriptional process.

Many bacterial mRNAs are regulated at the transcriptional or translational level by ligand-binding elements called riboswitches. Although they both bind adenine, the adenine riboswitches of Bacillus subtilis and Vibrio vulnificus differ by controlling transcription and translation, respectively. Here, we demonstrate that, beyond the obvious difference in transcriptional and translational modulation, both adenine riboswitches exhibit different ligand binding properties and appear to operate under different regulation regimes (kinetic versus thermodynamic). While the B. subtilis pbuE riboswitch fully depends on co-transcriptional binding of adenine to function, the V. vulnificus add riboswitch can bind to adenine after transcription is completed and still perform translation regulation. Further investigation demonstrates that the rate of transcription is critical for the B. subtilis pbuE riboswitch to perform efficiently, which is in agreement with a co-transcriptional regulation. Our results suggest that the nature of gene regulation control, that is transcription or translation, may have a high importance in riboswitch regulatory mechanisms.

Riboswitches are regulatory elements modulating gene expression in response to specific metabolite binding. It has been recently reported that riboswitch agonists may exhibit antimicrobial properties by binding to the riboswitch domain. Guanine riboswitches are involved in the regulation of transport and biosynthesis of purine metabolites, which are critical for the nucleotides cellular pool. Upon guanine binding, these riboswitches stabilize a 5′-untranslated mRNA structure that causes transcription attenuation of the downstream open reading frame. In principle, any agonistic compound targeting a guanine riboswitch could cause gene repression even when the cell is starved for guanine. Antibiotics binding to riboswitches provide novel antimicrobial compounds that can be rationally designed from riboswitch crystal structures. Using this, we have identified a pyrimidine compound (PC1) binding guanine riboswitches that shows bactericidal activity against a subgroup of bacterial species including well-known nosocomial pathogens. This selective bacterial killing is only achieved when guaA, a gene coding for a GMP synthetase, is under the control of the riboswitch. Among the bacterial strains tested, several clinical strains exhibiting multiple drug resistance were inhibited suggesting that PC1 targets a different metabolic pathway. As a proof of principle, we have used a mouse model to show a direct correlation between the administration of PC1 and the reduction of Staphylococcus aureus infection in mammary glands. This work establishes the possibility of using existing structural knowledge to design novel guanine riboswitch-targeting antibiotics as powerful and selective antimicrobial compounds. Particularly, the finding of this new guanine riboswitch target is crucial as community-acquired bacterial infections have recently started to emerge.

S-adenosylmethionine (SAM) riboswitches are widespread in bacteria, and up to five different SAM riboswitch families have been reported, highlighting the relevance of SAM regulation. On the basis of crystallographic and biochemical data, it has been postulated, but never demonstrated, that ligand recognition by SAM riboswitches involves key conformational changes in the RNA architecture. We show here that the aptamer follows a two-step hierarchical folding selectively induced by metal ions and ligand binding, each of them leading to the formation of one of the two helical stacks observed in the crystal structure. Moreover, we find that the anti-antiterminator P1 stem is rotated along its helical axis upon ligand binding, a mechanistic feature that could be common to other riboswitches. We also show that the nonconserved P4 helical domain is used as an auxiliary element to enhance the ligand-binding affinity. This work provides the first comprehensive characterization, to our knowledge, of a ligand-controlled riboswitch folding pathway.

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.gene regulation | RNA degradosome | translational control S ince the first demonstration that translation attenuation regulates the expression of the tryptophan operon (1), accumulating evidence has revealed the importance of posttranscriptional regulation in prokaryotes and eukaryotes alike. Posttranscriptional regulators include RNA molecules that operate through several mechanisms to control a wide range of physiological responses (2). Among newly identified RNA regulators are riboswitches, which are located in untranslated regions of several mRNAs and that modulate gene expression at the level of transcription, translation, or splicing (3). Riboswitches are highly structured regulatory domains that directly sense cellular metabolites such as amino acids, carbohydrates, coenzymes, and nucleobases. These genetic switches are composed of two modular domains consisting of an aptamer and an expression platform. The aptamer is involved in the specific recognition of the metabolite, and the expression platform is used to control gene expression by altering the structure of the mRNA. Recent bioinformatic analyses have reported the existence of several new RNA motifs exhibiting unconventional expression platforms (4, 5), suggesting that riboswitches using different regulation mechanisms are still likely to be discovered (6).The lysine riboswitch was first...

The VS ribozyme comprises ®ve helical segments (II±VI) in a formal H shape, organized by two threeway junctions. It interacts with its stem±loop substrate (I) by tertiary interactions. We have determined the global shape of the 3±4±5 junction (relating helices III±V) by electrophoresis and FRET. Estimation of the dihedral angle between helices II and V electrophoretically has allowed us to build a model for the global structure of the complete ribozyme. We propose that the substrate is docked into a cleft between helices II and VI, with its loop making a tertiary interaction with that of helix V. This is consistent with the dependence of activity on the length of helix III. The scissile phosphate is well placed to interact with the probable active site of the ribozyme, the loop containing A730.

Clostridium difficile is an anaerobic Gram-positive bacterium that causes intestinal infections with symptoms ranging from mild diarrhea to fulminant colitis. Cyclic diguanosine monophosphate (c-di-GMP) is a bacterial second messenger that typically regulates the switch from motile, free-living to sessile and multicellular behaviors in Gram-negative bacteria. Increased intracellular c-di-GMP concentration in C. difficile was recently shown to reduce flagellar motility and to increase cell aggregation. In this work, we investigated the role of the primary type IV pilus (T4P) locus in c-di-GMP-dependent cell aggregation. Inactivation of two T4P genes, pilA1 (CD3513) and pilB1 (CD3512), abolished pilus formation and significantly reduced cell aggregation under high c-di-GMP conditions. pilA1 is preceded by a putative c-di-GMP riboswitch, predicted to be transcriptionally active upon c-di-GMP binding. Consistent with our prediction, high intracellular c-di-GMP concentration increased transcript levels of T4P genes. In addition, single-round in vitro transcription assays confirmed that transcription downstream of the predicted transcription terminator was dose dependent and specific to c-di-GMP binding to the riboswitch aptamer. These results support a model in which T4P gene transcription is upregulated by c-di-GMP as a result of its binding to an upstream transcriptionally activating riboswitch, promoting cell aggregation in C. difficile. The Gram-positive spore-forming bacterium Clostridium difficile is the leading cause of nosocomial diarrhea and antibioticassociated colitis in hospital settings (1). Illness caused by C. difficile infections (CDI) may range from mild diarrhea to lifethreatening, fulminant colitis. Throughout its life cycle, C. difficile has to cope with multiple changing environments. In the early steps of CDI, in order to colonize the colon and produce toxins, C. difficile spores first germinate in the intestine in response to stimuli such as the presence of bile salts (2, 3). Vegetative cells need to reach the colon, likely attaching to and colonizing the gut mucosa, where they proliferate and produce toxins. Hence, C. difficile arguably needs to sense and integrate multiple environmental stimuli in order to coordinate the expression of its colonization and virulence factors during its journey through the gastrointestinal tract. The mechanisms allowing this adaptability in C. difficile have been the focus of many recent studies, including those on the agr quorum-sensing system, alternative sigma factors, two-component systems, the global transcription regulator CodY, and the sigma factor TcdR, which promotes TcdA and TcdB toxin expression (4-9).One way by which bacteria sense and respond to changes in their environment is through signal transduction involving secondary messenger molecules. The bacterial second messenger 3=-5= cyclic diguanosine monophosphate (c-di-GMP) plays multiple key roles in bacterial physiology and virulence (10-12). c-di-GMP has been shown to antagonistically control the...

The lysine riboswitch is associated to the lysC gene in Bacillus subtilis, and the binding of lysine modulates the RNA structure to allow the formation of an intrinsic terminator presumably involved in transcription attenuation. The complex secondary structure of the lysine riboswitch aptamer is organized around a five-way junction that undergoes structural changes upon ligand binding. Using single-round transcription assays, we show that a loop-loop interaction is important for lysine-induced termination of transcription. Moreover, upon close inspection of the secondary structure, we find that an unconventional kinkturn motif is present in one of the stems participating in the loop-loop interaction. We show that the K-turn adopts a pronounced kink and that it binds the K-turn-binding protein L7Ae of Archaeoglobus fulgidus in the low nanomolar range. The functional importance of this K-turn motif is revealed from single-round transcription assays, which show its importance for efficient transcription termination. This motif is essential for the loop-loop interaction, and consequently, for lysine binding. Taken together, our results depict for the first time the importance of a K-turn-dependent loop-loop interaction for the transcription regulation of a lysine riboswitch.