Riboswitch Class Research Articles

Riboswitch Scanner: an efficient pHMM-based web-server to detect riboswitches in genomic sequences.

Riboswitches are non-coding RNA located in the 5' untranslated regions where they bind a target metabolite used to specify the riboswitch class and control the expression of associated genes. Accurate identification of riboswitches is the first step towards understanding their regulatory and functional roles in the cell. In this article, we describe a new web application named Riboswitch Scanner which provides an automated pipeline for pHMM-based detection of riboswitches in partial as well as complete genomic sequences rapidly, with high sensitivity and specificity. Riboswitch Scanner can be freely accessed on the web at http://service.iiserkol.ac.in/∼riboscan/ mukherjee.sumit89@gmail.com Supplementary data are available at Bioinformatics online.

An Efficient Minimum Free Energy Structure-Based Search Method for Riboswitch Identification Based on Inverse RNA Folding.

Riboswitches are RNA genetic control elements that were originally discovered in bacteria and provide a unique mechanism of gene regulation. They work without the participation of proteins and are believed to represent ancient regulatory systems in the evolutionary timescale. One of the biggest challenges in riboswitch research is to find additional eukaryotic riboswitches since more than 20 riboswitch classes have been found in prokaryotes but only one class has been found in eukaryotes. Moreover, this single known class of eukaryotic riboswitch, namely the TPP riboswitch class, has been found in bacteria, archaea, fungi and plants but not in animals. The few examples of eukaryotic riboswitches were identified using sequence-based bioinformatics search methods such as a combination of BLAST and pattern matching techniques that incorporate base-pairing considerations. None of these approaches perform energy minimization structure predictions. There is a clear motivation to develop new bioinformatics methods, aside of the ongoing advances in covariance models, that will sample the sequence search space more flexibly using structural guidance while retaining the computational efficiency of sequence-based methods. We present a new energy minimization approach that transforms structure-based search into a sequence-based search, thereby enabling the utilization of well established sequence-based search utilities such as BLAST and FASTA. The transformation to sequence space is obtained by using an extended inverse RNA folding problem solver with sequence and structure constraints, available within RNAfbinv. Examples in applying the new method are presented for the purine and preQ1 riboswitches. The method is described in detail along with its findings in prokaryotes. Potential uses in finding novel eukaryotic riboswitches and optimizing pre-designed synthetic riboswitches based on ligand simulations are discussed. The method components are freely available for use.

Rational Re-engineering of a Transcriptional Silencing PreQ1 Riboswitch.

Re-engineered riboswitches that no longer respond to cellular metabolites, but that instead can be controlled by synthetic molecules, are potentially useful gene regulatory tools for use in synthetic biology and biotechnology fields. Previously, extensive genetic selection and screening approaches were employed to re-engineer a natural adenine riboswitch to create orthogonal ON-switches, enabling translational control of target gene expression in response to synthetic ligands. Here, we describe how a rational targeted approach was used to re-engineer the PreQ1 riboswitch from Bacillus subtilis into an orthogonal OFF-switch. In this case, the evaluation of just six synthetic compounds with seven riboswitch mutants led to the identification of an orthogonal riboswitch-ligand pairing that effectively repressed the transcription of selected genes in B. subtilis. The streamlining of the re-engineering approach, and its extension to a second class of riboswitches, provides a methodological platform for the creation of new orthogonal regulatory components for biotechnological applications including gene functional analysis and antimicrobial target validation and screening.

Secondary structural entropy in RNA switch (Riboswitch) identification.

BackgroundRNA regulatory elements play a significant role in gene regulation. Riboswitches, a widespread group of regulatory RNAs, are vital components of many bacterial genomes. These regulatory elements generally function by forming a ligand-induced alternative fold that controls access to ribosome binding sites or other regulatory sites in RNA. Riboswitch-mediated mechanisms are ubiquitous across bacterial genomes. A typical class of riboswitch has its own unique structural and biological complexity, making de novo riboswitch identification a formidable task. Traditionally, riboswitches have been identified through comparative genomics based on sequence and structural homology. The limitations of structural-homology-based approaches, coupled with the assumption that there is a great diversity of undiscovered riboswitches, suggests the need for alternative methods for riboswitch identification, possibly based on features intrinsic to their structure. As of yet, no such reliable method has been proposed.ResultsWe used structural entropy of riboswitch sequences as a measure of their secondary structural dynamics. Entropy values of a diverse set of riboswitches were compared to that of their mutants, their dinucleotide shuffles, and their reverse complement sequences under different stochastic context-free grammar folding models. Significance of our results was evaluated by comparison to other approaches, such as the base-pairing entropy and energy landscapes dynamics. Classifiers based on structural entropy optimized via sequence and structural features were devised as riboswitch identifiers and tested on Bacillus subtilis, Escherichia coli, and Synechococcus elongatus as an exploration of structural entropy based approaches. The unusually long untranslated region of the cotH in Bacillus subtilis, as well as upstream regions of certain genes, such as the sucC genes were associated with significant structural entropy values in genome-wide examinations.ConclusionsVarious tests show that there is in fact a relationship between higher structural entropy and the potential for the RNA sequence to have alternative structures, within the limitations of our methodology. This relationship, though modest, is consistent across various tests. Understanding the behavior of structural entropy as a fairly new feature for RNA conformational dynamics, however, may require extensive exploratory investigation both across RNA sequences and folding models.

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.

Going viral: riding the RNA wave to discovery.

Going viral: riding the RNA wave to discovery.

An Ancient Riboswitch Class in Bacteria Regulates Purine Biosynthesis and One-Carbon Metabolism

Over 30 years ago, ZTP (5-aminoimidazole-4-carboxamide riboside 5'-triphosphate), a modified purine biosynthetic intermediate, was proposed to signal 10-formyl-tetrahydrofolate (10f-THF) deficiency in bacteria. However, the mechanisms by which this putative alarmone or its precursor ZMP (5-aminoimidazole-4-carboxamide ribonucleotide, also known as AICAR) brings about any metabolic changes remain unexplained. Herein, we report the existence of a widespread riboswitch class that is most commonly associated with genes related to de novo purine biosynthesis and one-carbon metabolism. Biochemical data confirm that members of this riboswitch class selectively bind ZMP and ZTP with nanomolar affinity while strongly rejecting numerous natural analogs. Indeed, increases in the ZMP/ZTP pool, caused by folate stress in bacterial cells, trigger changes in the expression of a reporter gene fused to representative ZTP riboswitches in vivo. The wide distribution of this riboswitch class suggests that ZMP/ZTP signaling is important for species in numerous bacterial lineages.

Riboswitches as Potential Targets for Aminoglycosides Compared with rRNA Molecules: In Silico Study

Riboswitches are cis acting riboregulaters in non-coding region of the mRNAs. Their possible contribution in antibiotic targeting especially for FMN, TPP and lysine riboswitches in bacteria has been revealed since a decade ago. Regarding some studies on the possibility of the interaction between aminoglycosides and the artificial riboswitches, in this study we attempted to evaluate the binding potential of different types of aminoglycosides including gentamicin, amikacin, kanamycin, neomycin, tobramaycin, sisomicin and paromomycin with various classes of riboswitches using computational methods. Applying Auto Dock vina, it was shown that the binding energy of each kind of riboswitches with different types of aminoglycosides (riboswitches/aminoglycosides) is almost similar or sometimes more than the binding energy of the aminoglycoside with the corresponding binding cage of “16S rRNA A site” (16S rRNA A site/aminoglycosides) as aminoglycosides’ target site. The affinity between riboswitches and aminoglycosides is almost the same or higher than the affinity of riboswitches/natural ligands. In this study ampicillin was used as the negative control antibiotic and 5S rRNA was employed as the negative control RNA. Results showed that the binding energies of riboswitches/ampicillin and 5S rRNA/aminoglycosides are usually lower than the energy of riboswitches/aminoglycosides. Accordingly, lysine, glycine and SAM-I riboswitches were recognized as the best RNA targets for all of the aminoglycosides because of their higher binding energy. In the next step, docking results were further validated by rDock program. Furthermore, it was shown that hydrogen binding makes a key role in the binding energy between aminoglycosides and riboswitches. Moreover, MD simulation studies on lysine riboswitch/paromomycin complex confirmed the stability of the docked structure in the solvent containing magnesium and chloride ions.

Thermodynamic and kinetic folding of riboswitches.

Riboswitches are structured RNA regulatory elements located in the 5'-UTRs of mRNAs. Ligand-binding induces a structural rearrangement in these RNA elements, effecting events in downstream located coding sequences. Since they do not require proteins for their functions, they are ideally suited for computational analysis using the toolbox of RNA structure prediction methods. By their very definition riboswitch function depends on structural change. Methods that consider only the thermodynamic equilibrium of an RNA are therefore of limited use. Instead, one needs to employ computationally more expensive methods that consider the energy landscape and the folding dynamics on that landscape. Moreover, for the important class of kinetic riboswitches, the mechanism of riboswitch function can only be understood in the context of co-transcriptional folding. We present a computational approach to simulate the dynamic behavior of riboswitches during co-transcriptional folding in the presence and absence of a ligand. Our investigations show that the abstraction level of RNA secondary structure in combination with a dynamic folding landscape approach is expressive enough to understand how riboswitches perform their function. We apply our approach to a experimentally validated theophylline-binding riboswitch.

A riboswitch-containing sRNA controls gene expression by sequestration of a response regulator

The ethanolamine utilization (eut) locus of Enterococcus faecalis, containing at least 19 genes distributed over four polycistronic messenger RNAs, appears to be regulated by a single adenosyl cobalamine (AdoCbl)-responsive riboswitch. We report that the AdoCbl-binding riboswitch is part of a small, trans-acting RNA, EutX, which additionally contains a dual-hairpin substrate for the RNA binding-response regulator, EutV. In the absence of AdoCbl, EutX uses this structure to sequester EutV. EutV is known to regulate the eut messenger RNAs by binding dual-hairpin structures that overlap terminators and thus prevent transcription termination. In the presence of AdoCbl, EutV cannot bind to EutX and, instead, causes transcriptional read through of multiple eut genes. This work introduces riboswitch-mediated control of protein sequestration as a posttranscriptional mechanism to coordinately regulate gene expression.

Riboswitches. Sequestration of a two-component response regulator by a riboswitch-regulated noncoding RNA.

Riboswitches are ligand-binding elements contained within the 5' untranslated regions of bacterial transcripts, which generally regulate expression of downstream open reading frames. Here, we show that in Listeria monocytogenes, a riboswitch that binds vitamin B12 controls expression of a noncoding regulatory RNA, Rli55. Rli55, in turn, controls expression of the eut genes, whose products enable ethanolamine utilization and require B12 as a cofactor. Defects in ethanolamine utilization, or in its regulation by Rli55, significantly attenuate Listeria virulence in mice. Rli55 functions by sequestering the two-component response regulator EutV by means of a EutV-binding site contained within the RNA. Thus, Rli55 is a riboswitch-regulated member of the small group of regulatory RNAs that function by sequestering a protein and reveals a distinctive mechanism of signal integration in bacterial gene regulation.

A dual-action RNA switch for expression

Riboswitches are short segments of RNA that bind small molecules and switch between two different conformations, thereby regulating gene expression (see the Perspective by Chen and Gottesman). DebRoy et al. and Mellin et al. find a new class of riboswitches—in two different species of bacteria—that are both part of and regulate the production of a noncoding RNA. Each riboswitch ensures that a particular metabolic pathway is only activated in the presence of an essential small-molecule cofactor. In the absence of the cofactor, the full-length noncoding RNA is made and binds a regulator protein, preventing the regulator protein from inappropriately activating the metabolic pathway. S. DebRoy, M. Gebbie, A. Ramesh, J. R. Goodson, M. R. Cruz, A. van Hoof, W. C. Winkler, D. A. Garsin, A riboswitch-containing sRNA controls gene expression by sequestration of a response regulator. Science 345 , 937–940 (2014). [Abstract][Full Text] J. R. Mellin, M. Koutero, D. Dar, M.-A. Nahori, R. Sorek, P. Cossart, Sequestration of a two-component response regulator by a riboswitch-regulated noncoding RNA. Science 345 , 940–943 (2014). [Abstract][Full Text] J. Chen, S. Gottesman, Riboswitch regulates RNA. Science 345 , 876–877 (2014). [Abstract][Full Text]

Translational control by diverse RNA folds that recognize the metabolite preQ1

Typical paradigms of translational regulation utilize proteins, whereas riboswitches are economical, RNA-based elements that govern gene expression without protein partners. Present in all domains of life, riboswitches are commonly located in the 5´-untranslated regions of bacterial mRNAs where they establish feedback loops that respond to cellular levels of a specific small-molecule effector. Here we present the novel crystal structure of the recently discovered preQ1-III (class 3) riboswitch at 2.85 Å resolution. The 101-nucleotide switch features an internal loop pseudoknot (iPK) that coaxially stacks upon flanking helices P2 and P3. PreQ1(7-aminomethyl-7-deazaguanine) – the last soluble intermediate in the biosynthesis of the hypermodified tRNA base queuosine (Scheme I) – binds to form a major groove base triple of the form U·preQ1·C that utilizes highly conserved pyrimidine bases that help define this riboswitch class. This mode of effector binding involves non-canonical Watson-Crick pairing reminiscent of the phylogenetically unrelated preQ1-II (class 2) riboswitch. Remarkably, both riboswitch classes utilize dual U·A-U base triples that stack against the ligand. The respective nine-nucleotide motifs and their preQ1ligands superimpose with an rmsd of 0.4 Å; the class 2 and 3 structures are otherwise unrelated. The class 3 preQ1binding pocket also includes contributions from junctions that connect the P2-iPK-P3 coaxial stack with perpendicularly positioned helix P4. Of special interest is the observation that P4 can form a hairpin-loop pseudoknot (hPK) with the ribosome binding site (RBS) of the mRNA, suggesting a mode of ligand-dependent RBS sequestration. Biochemical experiments describing the preQ1dependence of hPK formation will be presented, as well as a comparison to known, translational class 1 and 2 preQ1riboswitch structures [1-3]. Implications for bacterial translational control will be discussed based on this diverse riboswitch family.

Structural, Functional, and Taxonomic Diversity of Three PreQ1 Riboswitch Classes

Previously, two riboswitch classes have been identified that sense and respond to the hypermodified nucleobase called prequeuosine1 (preQ1). The enormous expansion of available genomic DNA sequence data creates new opportunities to identify additional representatives of the known riboswitch classes and to discover novel classes. We conducted bioinformatics searches on microbial genomic DNA data sets to discover numerous additional examples belonging to the two previously known riboswitch classes for preQ1 (classes preQ1-I and preQ1-II), including some structural variants that further restrict ligand specificity. Additionally, we discovered a third preQ1-binding riboswitch class (preQ1-III) that is structurally distinct from previously known classes. These findings demonstrate that numerous organisms monitor the concentrations of this modified nucleobase by exploiting one or more riboswitch classes for this widespread compound.

Structure-Guided Design of Fluorescent S-Adenosylmethionine Analogs for a High-Throughput Screen to Target SAM-I Riboswitch RNAs

Many classes of S-adenosylmethionine (SAM)-binding RNAs and proteins are of interest as potential drug targets in diverse therapeutic areas, from infectious diseases to cancer. In the former case, the SAM-I riboswitch is an attractive target because this structured RNA element is found only in bacterial mRNAs and regulates multiple genes in several human pathogens. Here, we describe the synthesis of stable and fluorescent analogs of SAM in which the fluorophore is introduced through a functionalizable linker to the ribose. A Cy5-labeled SAM analog was shown to bind several SAM-I riboswitches via in-line probing and fluorescence polarization assays, including one from Staphylococcus aureus that controls the expression of SAM synthetase in this organism. A fluorescent ligand displacement assay was developed and validated for high-throughput screening of compounds to target the SAM-I riboswitch class.

ITC analysis of ligand binding to preQ₁ riboswitches.

Riboswitches regulate genes by binding to small-molecule effectors. Isothermal titration calorimetry (ITC) provides a label-free method to quantify the equilibrium association constant, K(A), of a riboswitch interaction with its cognate ligand. In addition to probing affinity and specific chemical contributions that contribute to binding, ITC can be used to measure the thermodynamic parameters of an interaction (ΔG, ΔH, and ΔS), in addition to the binding stoichiometry (N). Here, we describe methods developed to measure the binding affinity of various preQ1 riboswitch classes for the pyrrolopyrimidine effector, preQ1. Example isotherms are provided along with a review of various preQ1-II (class 2) riboswitch mutants that were interrogated by ITC to quantify the energetic contributions of specific interactions visualized in the crystal structure. Protocols for ITC are provided in sufficient detail that the reader can reproduce experiments independently, or develop derivative methods suitable for analyzing novel riboswitch-ligand binding interactions.

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. Intriguingly, 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 sub-nanomolar 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.

Evolutionary Evidence for Alternative Structure in RNA Sequence Co-variation

Sequence conservation and co-variation of base pairs are hallmarks of structured RNAs. For certain RNAs (e.g. riboswitches), a single sequence must adopt at least two alternative secondary structures to effectively regulate the message. If alternative secondary structures are important to the function of an RNA, we expect to observe evolutionary co-variation supporting multiple conformations. We set out to characterize the evolutionary co-variation supporting alternative conformations in riboswitches to determine the extent to which alternative secondary structures are conserved. We found strong co-variation support for the terminator, P1, and anti-terminator stems in the purine riboswitch by extending alignments to include terminator sequences. When we performed Boltzmann suboptimal sampling on purine riboswitch sequences with terminators we found that these sequences appear to have evolved to favor specific alternative conformations. We extended our analysis of co-variation to classic alignments of group I/II introns, tRNA, and other classes of riboswitches. In a majority of these RNAs, we found evolutionary evidence for alternative conformations that are compatible with the Boltzmann suboptimal ensemble. Our analyses suggest that alternative conformations are selected for and thus likely play functional roles in even the most structured of RNAs.

Structure of a class II preQ1 riboswitch reveals ligand recognition by a new fold

PreQ1 riboswitches regulate genes by binding the pyrrolopyrimidine intermediate preQ1 during biosynthesis of the essential tRNA base queuosine. We report the first preQ1-II riboswitch structure at 2.3 Å resolution, which uses a novel fold to achieve effector recognition at the confluence of a three-way-helical junction flanking a pseudoknotted ribosome-binding site (RBS). The results account for preQ1-II-riboswitch-mediated translational control, and expand the known repertoire of ligand binding modes utilized by regulatory RNAs.

One Platform, Five Brands: How Nature Cuts the Cost on Riboswitches

Riboswitches regulate gene expression in cis in response to changing metabolite concentrations in cells. Since flavin mononucleotide1,2 and thiamine pyrophosphate1,3 sensing riboswitches were discovered in 2002, more than 20 classes of metabolite-responsive riboswitches have been identified, primarily controlling essential metabolic or virulence genes in bacteria. Riboswitches are typically found in the mRNA 5′ untranslated region where they control expression usually by regulating premature transcription termination or translation initiation. Some riboswitches additionally control mRNA turnover4 or Rho-dependent termination.5 Regardless of their regulatory mechanism, the modular riboswitch structure is composed of an aptamer domain (ligand binding) and an effector domain (regulation). Ligand binding to the aptamer domain stabilizes an alternate structure and induces an effector domain switch to turn gene expression on or off. Riboswitches offer an efficient and rapid means to respond to changing metabolic conditions in a cell.