Lithium-sensing riboswitch classes regulate expression of bacterial cation transporter genes.

Lithium is rare in Earth's crust compared to the biologically relevant alkali metal cations sodium and potassium but can accumulate to toxic levels in some environments. We report the experimental validation of two distinct bacterial riboswitch classes that selectively activate gene expression in response to elevated Li+ concentrations. These RNAs commonly regulate the expression of nhaA genes coding for ion transporters that weakly discriminate between Na+ and Li+. Our findings demonstrated that the primary function of Li+ riboswitches and associated NhaA transporters is to prevent Li+ toxicity, particularly when bacteria are living at high pH. Additional riboswitch-associated genes revealed how some cells defend against the deleterious effects of Li+ in the biosphere, which might become more problematic as its industrial applications increase.

Architectures and complex functions of tandem riboswitches.

Riboswitch architectures that involve the binding of a single ligand to a single RNA aptamer domain result in ordinary dose-response curves that require approximately a 100-fold change in ligand concentration to cover nearly the full dynamic range for gene regulation. However, by using multiple riboswitches or aptamer domains in tandem, these ligand-sensing structures can produce additional, complex gene control outcomes. In the current study, we have computationally searched for tandem riboswitch architectures in bacteria to provide a more complete understanding of the diverse biological and biochemical functions of gene control elements that are made exclusively of RNA. Numerous different arrangements of tandem homologous riboswitch architectures are exploited by bacteria to create more 'digital' gene control devices, which operate over a narrower ligand concentration range. Also, two heterologous riboswitch aptamers are sometimes employed to create two-input Boolean logic gates with various types of genetic outputs. These findings illustrate the sophisticated genetic decisions that can be made by using molecular sensors and switches based only on RNA.

Na+ riboswitches regulate genes for diverse physiological processes in bacteria.

Organisms presumably have mechanisms to monitor and physiologically adapt to changes in cellular Na+ concentrations. Only a single bacterial protein has previously been demonstrated to selectively sense Na+ and regulate gene expression. Here we report a riboswitch class, previously called the 'DUF1646 motif', whose members selectively sense Na+ and regulate the expression of genes relevant to sodium biology. Many proteins encoded by Na+-riboswitch-regulated genes are annotated as metal ion transporters, whereas others are involved in mitigating osmotic stress or harnessing Na+ gradients for ATP production. Na+ riboswitches exhibit dissociation constants in the low mM range, and strongly reject all other alkali and alkaline earth ions. Likewise, only Na+ triggers riboswitch-mediated transcription and gene expression changes. These findings reveal that some bacteria use Na+ riboswitches to monitor, adjust and exploit Na+ concentrations and gradients, and in some instances collaborate with c-di-AMP riboswitches to coordinate gene expression during osmotic stress.

Riboswitches for the alarmone ppGpp expand the collection of RNA-based signaling systems.

Riboswitches are noncoding portions of certain mRNAs that bind metabolite, coenzyme, signaling molecule, or inorganic ion ligands and regulate gene expression. Most known riboswitches sense derivatives of RNA monomers. This bias in ligand chemical composition is consistent with the hypothesis that widespread riboswitch classes first emerged during the RNA World, which is proposed to have existed before proteins were present. Here we report the discovery and biochemical validation of a natural riboswitch class that selectively binds guanosine tetraphosphate (ppGpp), a widespread signaling molecule and bacterial "alarmone" derived from the ribonucleotide GTP. Riboswitches for ppGpp are predicted to regulate genes involved in branched-chain amino acid biosynthesis and transport, as well as other gene classes that previously had not been implicated to be part of its signaling network. This newfound riboswitch-alarmone partnership supports the hypothesis that prominent RNA World signaling pathways have been retained by modern cells to control key biological processes.

Tandem riboswitches form a natural Boolean logic gate to control purine metabolism in bacteria.

Gene control systems sometimes interpret multiple signals to set the expression levels of the genes they regulate. In rare instances, ligand-binding riboswitch aptamers form tandem arrangements to approximate the function of specific two-input Boolean logic gates. Here, we report the discovery of riboswitch aptamers for phosphoribosyl pyrophosphate (PRPP) that naturally exist either in singlet arrangements, or occur in tandem with guanine aptamers. Tandem guanine-PRPP aptamers can bind the target ligands, either independently or in combination, to approximate the function expected for an IMPLY Boolean logic gate to regulate transcription of messenger RNAs for de novo purine biosynthesis in bacteria. The existence of sophisticated all-RNA regulatory systems that sense two ancient ribonucleotide derivatives to control synthesis of RNA molecules supports the hypothesis that RNA World organisms could have managed a complex metabolic state without the assistance of protein regulatory factors.

Control of bacterial exoelectrogenesis by c-AMP-GMP.

Major changes in bacterial physiology including biofilm and spore formation involve signaling by the cyclic dinucleotides c-di-GMP and c-di-AMP. Recently, another second messenger dinucleotide, c-AMP-GMP, was found to control chemotaxis and colonization by Vibrio cholerae. We have identified a superregulon of genes controlled by c-AMP-GMP in numerous Deltaproteobacteria, including Geobacter species that use extracellular insoluble metal oxides as terminal electron acceptors. This exoelectrogenic process has been studied for its possible utility in energy production and bioremediation. Many genes involved in adhesion, pilin formation, and others that are important for exoelectrogenesis are controlled by members of a variant riboswitch class that selectively bind c-AMP-GMP. These RNAs constitute, to our knowledge, the first known specific receptors for c-AMP-GMP and reveal that this molecule is used by many bacteria to control specialized physiological processes.

Riboswitches in eubacteria sense the second messenger c-di-AMP.

Cyclic di-adenosine monophosphate (c-di-AMP) is a recently discovered bacterial second messenger implicated in the control of cell wall metabolism, osmotic stress responses and sporulation. However, the mechanisms by which c-di-AMP triggers these physiological responses have remained largely unknown. Notably, a candidate riboswitch class called ydaO associates with numerous genes involved in these same processes. Although a representative ydaO motif RNA recently was reported to weakly bind ATP, we report that numerous members of this noncoding RNA class selectively respond to c-di-AMP with subnanomolar affinity. Our findings resolve the mystery regarding the primary ligand for this extremely common riboswitch class and expose a major portion of the super-regulon of genes that are controlled by the widespread bacterial second messenger c-di-AMP.

Identification of ligand analogues that control c-di-GMP riboswitches.

Riboswitches for the bacterial second messenger c-di-GMP control the expression of genes involved in numerous cellular processes such as virulence, competence, biofilm formation, and flagella synthesis. Therefore, the two known c-di-GMP riboswitch classes represent promising targets for developing novel modulators of bacterial physiology. Here, we examine the binding characteristics of circular and linear c-di-GMP analogues for representatives of both class I and II c-di-GMP riboswitches derived from the pathogenic bacterium Vibrio choleae (class I) and Clostridium difficile (class II). Some compounds exhibit values for apparent dissociation constant (K(D)) below 1 µM and associate with riboswitch RNAs during transcription with a speed that is sufficient to influence riboswitch function. These findings are consistent with the published structural models for these riboswitches and suggest that large modifications at various positions on the ligand can be made to create novel compounds that target c-di-GMP riboswitches. Moreover, we demonstrate the potential of an engineered allosteric ribozyme for the rapid screening of chemical libraries for compounds that bind c-di-GMP riboswitches.

Widespread genetic switches and toxicity resistance proteins for fluoride.

Most riboswitches are metabolite-binding RNA structures located in bacterial messenger RNAs where they control gene expression. We have discovered a riboswitch class in many bacterial and archaeal species whose members are selectively triggered by fluoride but reject other small anions, including chloride. These fluoride riboswitches activate expression of genes that encode putative fluoride transporters, enzymes that are known to be inhibited by fluoride, and additional proteins of unknown function. Our findings indicate that most organisms are naturally exposed to toxic levels of fluoride and that many species use fluoride-sensing RNAs to control the expression of proteins that alleviate the deleterious effects of this anion.

Mechanism for gene control by a natural allosteric group I ribozyme.

An allosteric ribozyme consisting of a metabolite-sensing riboswitch and a group I self-splicing ribozyme was recently found in the pathogenic bacterium Clostridium difficile. The riboswitch senses the bacterial second messenger c-di-GMP, thereby controlling 5'-splice site choice by the downstream ribozyme. The proximity of this allosteric ribozyme to the open reading frame (ORF) for CD3246 suggests that coenzyme-mediated regulation of splicing controls expression of this putative virulence gene. In the presence of c-di-GMP, the allosteric ribozyme in the CD3246 precursor transcript generates a spliced transcript that retains the riboswitch aptamer. In the absence of c-di-GMP, the ribozyme mediates an alternative GTP attack that results in a truncated transcript (alternative GTP-attack product). Using reporter assays in Escherichia coli, we investigated the difference in gene expression between the spliced product and the alternative GTP-attack product. We provide evidence that CD3246 gene expression is activated if allosteric ribozyme splicing creates a ribosome binding site (RBS) for translation from a UUG start codon. In addition, biochemical and genetic analyses reveal that the riboswitch may further control CD3246 expression by revealing or occluding this newly formed RBS. Therefore, this architecture provides the riboswitch with a mechanism for extended regulation after splicing has occurred or as a backup mechanism for suppression of translation in the event of misregulated splicing.

Challenges of ligand identification for riboswitch candidates.

Expanding DNA sequence databases and improving methods for comparative analysis are being exploited to identify numerous noncoding RNA elements including riboswitches. Ligands for many riboswitch classes usually can be inferred based on the genomic contexts of representative RNAs, and complex formation or genetic regulation subsequently demonstrated experimentally. However, there are several candidate riboswitches for which ligands have not been identified. In this report, we discuss three of the most compelling riboswitch candidates: the ykkC/ykkD, yybP/ykoY and pfl RNAs. Each of these RNAs is numerous, phylogenetically widespread, and carries features that are hallmarks of metabolite-binding riboswitches, such as a well-conserved aptamer-like structure and apparent interactions with gene regulation elements such as ribosome binding sites or intrinsic transcription termination stems. These RNAs likely represent only a small sampling of the challenging motifs that researchers will encounter as new noncoding RNAs are identified.

An allosteric self-splicing ribozyme triggered by a bacterial second messenger.

Group I self-splicing ribozymes commonly function as components of selfish mobile genetic elements. We identified an allosteric group I ribozyme, wherein self-splicing is regulated by a distinct riboswitch class that senses the bacterial second messenger c-di-GMP. The tandem RNA sensory system resides in the 5' untranslated region of the messenger RNA for a putative virulence gene in the pathogenic bacterium Clostridium difficile. c-di-GMP binding by the riboswitch induces folding changes at atypical splice site junctions to modulate alternative RNA processing. Our findings indicate that some self-splicing ribozymes are not selfish elements but are harnessed by cells as metabolite sensors and genetic regulators.

Identification of 22 candidate structured RNAs in bacteria using the CMfinder comparative genomics pipeline.

We applied a computational pipeline based on comparative genomics to bacteria, and identified 22 novel candidate RNA motifs. We predicted six to be riboswitches, which are mRNA elements that regulate gene expression on binding a specific metabolite. In separate studies, we confirmed that two of these are novel riboswitches. Three other riboswitch candidates are upstream of either a putative transporter gene in the order Lactobacillales, citric acid cycle genes in Burkholderiales or molybdenum cofactor biosynthesis genes in several phyla. The remaining riboswitch candidate, the widespread Genes for the Environment, for Membranes and for Motility (GEMM) motif, is associated with genes important for natural competence in Vibrio cholerae and the use of metal ions as electron acceptors in Geobacter sulfurreducens. Among the other motifs, one has a genetic distribution similar to a previously published candidate riboswitch, ykkC/yxkD, but has a different structure. We identified possible non-coding RNAs in five phyla, and several additional cis-regulatory RNAs, including one in epsilon-proteobacteria (upstream of purD, involved in purine biosynthesis), and one in Cyanobacteria (within an ATP synthase operon). These candidate RNAs add to the growing list of RNA motifs involved in multiple cellular processes, and suggest that many additional RNAs remain to be discovered.

Control of alternative RNA splicing and gene expression by eukaryotic riboswitches.

Bacteria make extensive use of riboswitches to sense metabolites and control gene expression, and typically do so by modulating premature transcription termination or translation initiation. The most widespread riboswitch class known in bacteria responds to the coenzyme thiamine pyrophosphate (TPP), which is a derivative of vitamin B1. Representatives of this class have also been identified in fungi and plants, where they are predicted to control messenger RNA splicing or processing. We examined three TPP riboswitches in the filamentous fungus Neurospora crassa, and found that one activates and two repress gene expression by controlling mRNA splicing. A detailed mechanism involving riboswitch-mediated base-pairing changes and alternative splicing control was elucidated for precursor NMT1 mRNAs, which code for a protein involved in TPP metabolism. These results demonstrate that eukaryotic cells employ metabolite-binding RNAs to regulate RNA splicing events that are important for the control of key biochemical processes.

Antibacterial lysine analogs that target lysine riboswitches.

Lysine riboswitches are bacterial RNA structures that sense the concentration of lysine and regulate the expression of lysine biosynthesis and transport genes. Members of this riboswitch class are found in the 5' untranslated region of messenger RNAs, where they form highly selective receptors for lysine. Lysine binding to the receptor stabilizes an mRNA tertiary structure that, in most cases, causes transcription termination before the adjacent open reading frame can be expressed. A lysine riboswitch conceivably could be targeted for antibacterial therapy by designing new compounds that bind the riboswitch and suppress lysine biosynthesis and transport genes. As a test of this strategy, we have identified several lysine analogs that bind to riboswitches in vitro and inhibit Bacillus subtilis growth, probably through a mechanism of riboswitch-mediated repression of lysine biosynthesis. These results indicate that riboswitches could serve as new classes of antibacterial drug targets.

Tandem riboswitch architectures exhibit complex gene control functions.

Riboswitches are structured RNAs typically located in the 5' untranslated regions of bacterial mRNAs that bind metabolites and control gene expression. Most riboswitches sense one metabolite and function as simple genetic switches. However, we found that the 5' region of the Bacillus clausii metE messenger RNA includes two riboswitches that respond to S-adenosylmethionine and coenzyme B12. This tandem arrangement yields a composite gene control system that functions as a two-input Boolean NOR logic gate. These findings and the discovery of additional tandem riboswitch architectures reveal how simple RNA elements can be assembled to make sophisticated genetic decisions without involving protein factors.

Thiamine pyrophosphate riboswitches are targets for the antimicrobial compound pyrithiamine.

Thiamine metabolism genes are regulated in numerous bacteria by a riboswitch class that binds the coenzyme thiamine pyrophosphate (TPP). We demonstrate that the antimicrobial action of the thiamine analog pyrithiamine (PT) is mediated by interaction with TPP riboswitches in bacteria and fungi. For example, pyrithiamine pyrophosphate (PTPP) binds the TPP riboswitch controlling the tenA operon in Bacillus subtilis. Expression of a TPP riboswitch-regulated reporter gene is reduced in transgenic B. subtilis or Escherichia coli when grown in the presence of thiamine or PT, while mutant riboswitches in these organisms are unresponsive to these ligands. Bacteria selected for PT resistance bear specific mutations that disrupt ligand binding to TPP riboswitches and derepress certain TPP metabolic genes. Our findings demonstrate that riboswitches can serve as antimicrobial drug targets and expand our understanding of thiamine metabolism in bacteria.

6S RNA is a widespread regulator of eubacterial RNA polymerase that resembles an open promoter.

6S RNA is an abundant noncoding RNA in Escherichia coli that binds to sigma70 RNA polymerase holoenzyme to globally regulate gene expression in response to the shift from exponential growth to stationary phase. We have computationally identified >100 new 6S RNA homologs in diverse eubacterial lineages. Two abundant Bacillus subtilis RNAs of unknown function (BsrA and BsrB) and cyanobacterial 6Sa RNAs are now recognized as 6S homologs. Structural probing of E. coli 6S RNA and a B. subtilis homolog supports a common secondary structure derived from comparative sequence analysis. The conserved features of 6S RNA suggest that it binds RNA polymerase by mimicking the structure of DNA template in an open promoter complex. Interestingly, the two B. subtilis 6S RNAs are discoordinately expressed during growth, and many proteobacterial 6S RNAs could be cotranscribed with downstream homologs of the E. coli ygfA gene encoding a putative methenyltetrahydrofolate synthetase. The prevalence and robust expression of 6S RNAs emphasize their critical role in bacterial adaptation.

New RNA motifs suggest an expanded scope for riboswitches in bacterial genetic control.

The expression of certain genes involved in fundamental metabolism is regulated by metabolite-binding "riboswitch" elements embedded within their corresponding mRNAs. We have identified at least six additional elements within the Bacillus subtilis genome that exhibit characteristics of riboswitch function (glmS, gcvT, ydaO/yuaA, ykkC/yxkD, ykoK, and yybP/ykoY). These motifs exhibit extensive sequence and secondary-structure conservation among many bacterial species and occur upstream of related genes. The element located upstream of the glmS gene in Gram-positive organisms functions as a metabolite-dependent ribozyme that responds to glucosamine-6-phosphate. Other motifs form complex folded structures when transcribed as RNA molecules and carry intrinsic terminator structures. These findings indicate that riboswitches serve as a major genetic regulatory mechanism for the control of metabolic genes in many microbial species.

An mRNA structure in bacteria that controls gene expression by binding lysine.

Riboswitches are metabolite-responsive genetic control elements that reside in the untranslated regions (UTRs) of certain messenger RNAs. Herein, we report that the 5'-UTR of the lysC gene of Bacillus subtilis carries a conserved RNA element that serves as a lysine-responsive riboswitch. The ligand-binding domain of the riboswitch binds to L-lysine with an apparent dissociation constant (KD) of approximately 1 micro M, and exhibits a high level of molecular discrimination against closely related analogs, including D-lysine and ornithine. Furthermore, we provide evidence that this widespread class of riboswitches serves as a target for the antimetabolite S-(2-aminoethyl)-L-cysteine (AEC). These findings add support to the hypotheses that direct sensing of metabolites by messenger RNAs is a fundamental form of genetic control and that riboswitches represent a new class of antimicrobial drug targets.