Biol.

Cell (2008) 100, 111 (Printed in Great Britain)

doi:10.1042/BC20070088

Review

Purine sensing by riboswitches

Jane N. Kim* and Ronald R. Breaker*1
*Department of Molecular, Cellular and Developmental Biology, Yale University, P.O. Box 208103, New Haven, CT 06520-8103, U.S.A., Department of Molecular Biophysics and Biochemistry, Yale University, P.O. Box 208103, New Haven, CT 06520-8103, U.S.A., and Howard Hughes Medical Institute, Yale University, P.O. Box 208103, New Haven, CT 06520-8103, U.S.A.

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Structured mRNA elements called riboswitches control gene expression by binding to small metabolites. Over a dozen riboswitch classes have been characterized that target a broad range of molecules and vary widely in size and secondary structure. Four of the known riboswitch classes recognize purines or modied purines. Three of these classes are closely related in conserved sequence and secondary structure, but members of these classes selectively recognize guanine, adenine or 2 -deoxyguanosine. Members of the fourth riboswitch class adopt a distinct structure to form a selective binding pocket for the guanine analogue preQ1 (7-aminomethyl7-deazaguanine). All four classes of purine-sensing riboswitches are most likely to recognize their respective metabolites by utilizing a riboswitch residue to make a canonical WatsonCrick base-pair with the ligand. This review will provide a summary of the purine-sensing riboswitches, as well as discuss the complex functions and applications of these RNAs.

Biology of the Cell

Introduction Riboswitches are gene control elements primarily found in the 5 UTRs (untranslated regions) of bacterial mRNAs (Mandal and Breaker, 2004a; Nudler and Mironov, 2004; Winkler and Breaker, 2005). These structured RNA motifs bind to specic ligands, which serves to regulate expression of the adjacent gene or set of genes. Riboswitch-mediated gene control can function as a feedback mechanism, since the targeted ligand is often synthesized or processed by the gene(s) located downstream of the riboswitch. Riboswitches generally consist of two regions. One is called an aptamer, which is a conserved and structured receptor that binds a specic ligand. Before the rst riboswitch was characterized, a variety of RNA aptamers were engineered using in vitro evolution that recognize a wide range of small molecules (Gold et al., 1995; Osborne and Ellington, 1997). The discovery of riboswitches revealed that the ability of RNA to form precision binding pockets for small molecules is harnessed by modern cells to sense metabolites. The second riboswitch region is called an

expression platform, which is responsible for modulating gene expression as a result of ligand binding. In bacteria, expression platforms typically control transcription (Figure 1A) by forming a terminator or antiterminator stem, or control translation (Figure 1B) by either occluding or exposing the ShineDalgarno sequence that needs to be recognized by the ribosome during translation initiation. Riboswitch-mediated gene control is prevalent in bacteria, regulating approx. 2% of all genes in the bacterial species Bacillus subtilis (Mandal et al., 2003). To date, more than a dozen riboswitch classes have been identied, as well as many candidate riboswitch classes (Barrick et al., 2004; Corbino et al., 2005; Weinberg et al., 2007). Although most of these riboswitches have been found exclusively in bacterial species, thiamine-pyrophosphate-sensing riboswitches have also been identied in plants and fungi (Kubodera et al., 2003; Sudarsan et al., 2003a; Galagan et al., 2005, Cheah et al., 2007), and other riboswitches may also be present in eukaryotes (Borsuk et al., 2007). Although few examples of riboswitches have been identied in eukaryotes to date,
In vitro evolution: A phrase designating methods used to create functional RNA, DNA or proteins from vast pools of mutant molecules.
ShineDalgarno sequence: The short purine-rich sequence that is approx. 10 nt upstream of a bacterial mRNA start codon, which is recognized by the ribosome during translation initiation.

1 To

whom correspondence should be addressed (email ronald.breaker@yale.edu). Key words: adenine, allosteric RNA, aptamer, guanine, riboswitch, RNA switch. Abbreviations used: 3D, three-dimensional; 2,6-DAP, 2,6-diaminopurine; dG, 2 -deoxyguanosine; preQ1 , 7-aminomethyl-7-deazaguanine; UTR, untranslated region.

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Figure 1 Riboswitch-mediated gene control mechanisms (A) Transcription regulation by riboswitches. When the riboswitch ligand is unavailable, transcription of the downstream gene is permitted due to the formation of an anti-terminator stem. However, when the aptamer is able to bind the ligand, the anti-terminator is unable to assemble, and transcription termination occurs via formation of a terminator stem. (B) Translation regulation by riboswitches. In the absence of ligand, a ribosome binds to the RBS (ribosome-binding site) on the mRNA and initiates translation. When the ligand is available, the RBS becomes sequestered and is not recognized by the ribosome, thus translation cannot occur.

the large amount of non-coding RNAs that are transcribed in these cells provides enormous potential for metaboliteRNA interactions. The known purine-sensing riboswitches can be assigned to one of four riboswitch classes (Mandal et al., 2003; Mandal and Breaker, 2004b; Kim et al., 2007; Roth et al., 2007). However, it is possible that there are more purine-sensing riboswitch classes that have yet to be discovered. In this regard it is notable that a wide range of engineered RNA aptamers have been isolated that recognize a variety of purines, such as guanine and xanthine (Kiga et al., 1998), and purine-related molecules, such as GTP (Davis and Szostak, 2002) and cAMP (Koizumi and Breaker, 2000). These engineered aptamers for purines and purine derivatives are distinct from those identied from natural sources. Thus RNA has the ability to form a great diversity of receptors that bind various purines and purine-like compounds, indicating that additional distinct riboswitch classes for these compounds probably remain to be identied. It should also be noted that there are other riboswitch classes that target purine-containing compounds, but are not included in the purine-sensing

riboswitch group described in this review. These motifs include the coenzyme B12 riboswitch class (Nahvi et al., 2002), the various S-adenosylmethionine riboswitch classes (Winkler et al., 2003; Corbino et al., 2005; Fuchs et al., 2006; Weinberg et al., 2007) and the S-adenosylhomocysteine riboswitch class (J. X. Wang, E. R. Lee, D. Rivera-Morales, J. Lim and R. R. Breaker, unpublished data). However, the purine moiety constitutes only a portion of these ligands, whereas the four described here that are labelled as purine-sensing riboswitches all recognize a purine, modied purine or a purine deoxyribonucleoside. Also, members of the purine-sensing riboswitch classes all appear to bind their respective metabolites, at least in part by making a WatsonCrick base-pair with their target ligand.

Anatomies and functions of the purine-sensing riboswitches

Guanine and adenine riboswitches carry similar aptamers that recognize distinct ligands

A number of the riboswitches characterized initially were found by examining literature that describes the

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Review
guanine riboswitches to a uridine residue in adenine riboswitches. It was suspected that this nucleotide is critical in targeting the ligand, probably by making a WatsonCrick base-pair with the ligand. Indeed, a single nucleotide change between C and U residues was found to be the only alteration necessary to change ligand specicity between guanine and adenine (Mandal and Breaker, 2004b). The precise interactions used by guanine and adenine riboswitch aptamers to selectively bind their ligands were conrmed and expanded through the use of X-ray crystallization and NMR analyses. For example, X-ray crystallographic studies of the guanine riboswitch upstream of xptpbuX in B. subtilis (Batey et al., 2004; Serganov et al., 2004) and the adenine riboswitch from the add gene of Vibrio vulnicus (Serganov et al., 2004) bound to their respective ligands revealed the details of purine binding by these RNAs. The 3D (three-dimensional) structures of the two aptamers are virtually identical, and the RNAs are structured so that they almost completely envelop the ligands. The global architecture of these aptamers explains some of the nucleotide sequences and secondary structures that are characteristic of guanine and adenine riboswitches. For example, the P2 and P3 stems (Figures 2A and 2B) adopt a parallel alignment and are held in place by interactions between the L2 and L3 loops. When the metabolite is bound, the overall structure of the aptamer is somewhat similar to that of a tuning fork (Batey et al., 2004). The structural requirements place length restrictions on the P2 and P3 stems, each of which are usually formed from 7 bp. Furthermore, the nucleotides in the loops that form the tertiary contacts are highly conserved (Mandal et al., 2003), and have proven to be necessary for ligand binding (Lemay et al., 2006). Several other studies have been conducted that further support the structural models for guanine and adenine aptamers (Gilbert et al., 2006a; Lemay and Lafontaine, 2007). Results from X-ray crystallization studies (Batey et al., 2004; Serganov et al., 2004) and from NMR analysis (Noeske et al., 2005) conrmed that a cytidine residue makes a WatsonCrick base-pair with the guanine ligand (Figure 2A). In contrast, the 3D

involvement of small molecule metabolites as regulators of gene control. In some instances, researchers reported the identication of a particular metabolite as a regulator of gene expression, although a regulatory protein that might respond to changes in metabolite concentration notably remained undiscovered. In each of these cases, gene regulation was later found to be riboswitch mediated, meaning that the mRNA under control directly bound its regulatory metabolite in the absence of proteins and that the aptamer domain is an essential component of gene regulation. For example, guanine was shown to repress expression of the xptpbuX operon, which consists of genes that transport and metabolize purines in B. subtilis (Christiansen et al., 1997). However, a protein factor that could sense guanine and control operon expression had not been identied. By comparing the nucleotide sequences of the xptpbuX 5 UTR with regions upstream of other purine metabolism genes, a highly conserved region was identied that forms the aptamer for a class of riboswitches that selectively binds to guanine (Mandal et al., 2003). Subsequently, numerous guanine riboswitch representatives were identied in various bacterial species, always located upstream of a gene or operon involved in purine synthesis or transport. Assays such as in-line probing (Soukup and Breaker, 1999) and equilibrium dialysis conrmed that guanine could bind to the riboswitch (Figure 2A) and promote formation of a transcription terminator stem. The K d of guanine binding to the aptamer is approx. 5 nM, and this riboswitch selectively binds guanine and rejects adenine and many other guanine analogues. Soon after the guanine riboswitch class was identied, a subset of this class was found to recognize adenine (Mandal et al., 2003; Mandal and Breaker, 2004b). Representatives of adenine-sensing riboswitches reside in the 5 UTRs of mRNAs encoding adenine metabolism and transport proteins, and one example has been shown to bind adenine with a K d of approx. 300 nM. Adenine riboswitch aptamers closely resemble guanine riboswitch aptamers in secondary structure and conserved sequence (Figure 2B). However, a highly-conserved nucleotide within the aptamer core is changed from a cytidine residue in

In-line probing: An RNA structure probing assay that takes advantage of the natural instability of RNA to assess the local structural dynamics of a polynucleotide chain.

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Figure 2 Secondary structures and partial 3D structure views of purine-sensing riboswitch aptamers (A) Sequence and secondary structure of a guanine-sensing aptamer of the xptpbuX riboswitch from B. subtilis (Mandal et al., 2003). The residue making a WatsonCrick base-pair with the ligand is highlighted in blue, and the additional nucleotides that directly contact the ligand are indicated with Roman numerals. A 3D view of the ligand-binding pocket bound to its ligand is depicted on the right-hand side (PDB code 1y27). Broken lines indicate hydrogen-bonding interactions. The nucleotide that makes a canonical base-pair with the ligand is labelled in blue, and the surrounding residues are numbered to match with

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their corresponding positions in the secondary structure model. (B) Sequence and secondary structure of an adenine-sensing aptamer of the add riboswitch from V. vulnicus (Mandal et al., 2003). The ligand-binding pocket is depicted at the right-hand side (PDB code 1y26). Other details are as described in (A). (C) Sequence and secondary structure of the dG-sensing riboswitch aptamer motif I-A from M. orum. The 3D structure of guanine bound to the xpt aptamer (PDB code 1y27) is shown, indicating that three of the four key aptamer nucleotides that contact the ligand are mutated (red). The arrow points to the location of attachment for a deoxyribose moiety to the purine ring of guanine. Other details are as described in (A). (D) Sequence and secondary structure of the queC riboswitch aptamer from B. subtilis, which recognizes preQ1 .

structure of the add motif shows a uridine residue in this position, which creates a canonical base-pair with the adenine metabolite (Figure 2B). However, these nucleotides are not the only riboswitch residues involved in ligand recognition. Many of the conserved residues in the aptamer junctions make up the conserved core region, which creates the tight binding pocket for the ligand, and a total of four residues within this region are involved in making direct ligand contacts.

Variants of the guanine riboswitch class sense dG (2 -deoxyguanosine)

RNA motifs similar to the guanine and adenine riboswitches were recently identied in Mesoplasma orum (Kim et al., 2007). The aptamer regions and predicted secondary structures are similar to those of the guanine and adenine riboswitches, whereas the expression platforms resemble transcription terminator stems. However, these RNA elements possess nucleotides that vary from the core consensus sequence of the guanine and adenine riboswitches. Given that these changes to the core of the aptamer were known to be important for ligand binding, we speculated that these RNAs likely would exhibit altered ligand binding specicity. Such demonstrations of altered ligand specicity have been reported previously for engineered RNA aptamers. For example, an aptamer that targets citrulline was altered to sense arginine by changing only 3 out of a total of 44 nucleotides (Famulok, 1994). X-ray crystallography of both the citrulline and arginine aptamers revealed that their folded structures were virtually identical (Yang, et al., 1996), despite the changes that altered ligand specicity. A similar effect is also likely occurring for aptamers that preferentially bind 3-methylxanthine after acquiring a

single mutation compared with a parent aptamer for theophylline (Soukup et al., 2000). As predicted, some of the guanine riboswitch variants identied in M. orum exhibit altered ligandbinding specicity and afnity. Most interesting are several that selectively bind the purine-containing nucleoside dG. One example (motif IA; Figure 2C) is located upstream of a ribonucleotide reductase gene, which encodes an enzyme that converts phosphorylated ribonucleotides into their corresponding phosphorylated deoxyribonucleotides (Thelander and Reichard, 1979; Kolberg et al., 2004). In-line probing assays and transcription termination experiments in vitro determined that motif IA binds dG (K d , approx. 80 nM) and discriminates by nearly 3 orders of magnitude against guanosine (K d , approx. 5 M), which is the RNA version of this nucleoside. In contrast, group I self-splicing ribozymes undergo processing when bound to guanosine, but reject dG as a substrate (Cech et al., 1981; Kruger et al., 1982). Additional natural RNAs might exist that are capable of responding precisely to nucleosides and nucleotides, as opposed to free purine bases. For example, a viral RNA has been suggested to sense ATP (Shu and Guo, 2003), although details of this putative interaction have yet to be reported. The core consensus sequence variations from the known guanine riboswitches are thought to be important in determining ligand specicity. Due to the high selectivity for dG in vitro, these newfound riboswitches are predicted to target dG in vivo. Because the guanine and dG aptamers resemble one another, it was postulated that motif IA makes a canonical basepair with the guanine moiety of dG. A single C-to-U mutation was made at the nucleotide corresponding to the residue in guanine and adenine aptamers that are known to form a WatsonCrick base-pair with the ligand. The wild-type motif IA recognizes dG

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with an afnity over 1 103 -fold greater than that exhibited by the mutant RNA carrying this C-to-U mutation. In comparison, 2 -deoxyadenosine is not sensed by the wild-type aptamer or by the C-to-U mutant. However, whereas the wild-type motif IA normally does not bind 2,6-DAP (2,6-diaminopurine) 2 -deoxyribonucleoside, the mutant construct recognizes this ligand with a K d of 8 M. 2,6-DAP is a closely related analogue of adenine that carries an additional amino group at the C2 position of the purine ring. Interestingly, it was previously shown that an adenine riboswitch recognizes 2,6-DAP with an approx. 30-fold greater afnity than that for adenine (Mandal and Breaker, 2004b). Examination of the crystal structure of adenine bound to the add motif reveals that the amino group of 2,6-DAP can make two extra hydrogen bonds with the aptamer, strengthening the binding interaction between riboswitch and ligand (Serganov et al., 2004). Thus it is likely that 2,6-DAP 2 -deoxyriboside is bound more tightly than 2 -deoxyadenosine by the C-toU mutated dG riboswitch due to the formation of an additional hydrogen bond, as observed with the analogous guanine riboswitch mutant. Given the similar structural and functional characteristics, the dG riboswitch class probably makes a WatsonCrick basepair with the guanine moiety of its ligand using the same pairing interactions used by guanine- and adenine-binding riboswitches. However, the three other nucleotides that directly contact the ligand in guanine and adenine riboswitches are mutated in the dG riboswitch representatives (Figure 2C). This shows the core of a guanine aptamer binding site, where the red nucleotides are mutated in dG aptamers, presumably to selectively recognize the deoxyribose ring.
PreQ1 (7-aminomethyl-7-deazaguanine) riboswitches are small, but selective

and one example binds preQ1 with a K d of approx. 50 nM. This binding event is predicted to cause transcription termination in approx. 40% of the known representatives, as shown by the existence of terminator stems in these examples. The remaining representatives appear to control translation. The preQ1 riboswitch class structure does not resemble that of the other known purine-binding motifs, but consists of one or two simple stemloop regions, with a minimum aptamer length of only 34 nt. This small and simple riboswitch is, nevertheless, selective for preQ1 over related analogues, such as guanine and hypoxanthine. The modication to the purine ring that makes preQ1 distinct from guanine occurs at the 7 position of the ring. Therefore, preQ1 retains the ability to form a canonical base-pair with cytidine. Again, it was postulated that a conserved cytidine residue within the preQ1 aptamer might be responsible for preQ1 recognition, as has been observed with other purine riboswitch aptamers. Indeed, when a universally conserved C residue at the base of P1 was mutated to a U residue, the riboswitch no longer binds preQ1 , but 2,6-DAP (an adenine-related molecule) is recognized. Thus, although the secondary structure model of the preQ1 riboswitch differs from that of the other purine riboswitches, recognition of the ligand is still likely to be achieved via WatsonCrick base-pairing to a specic cytidine residue within the riboswitch aptamer.

A riboswitch class was identied recently that recognizes the modied purine preQ1 (Roth et al., 2007). This molecule is a precursor used for the biosynthesis of queuosine, a hypermodied 7-deazaguanosine nucleoside that is found in the anticodon wobble position of certain tRNAs (Harada and Nishimura, 1972). This RNA motif (Figure 2D) is most often found upstream of genes involved in queuosine biosynthesis,

Complex functions and applications of purine-sensing riboswitches Riboswitches are not always only simple genetic control switches that turn on or off in the presence of a particular ligand concentration, but some can operate with more complex functions. This is true of some members of the purine-sensing riboswitches, and detailed studies have been performed examining the complex functions of these riboswitches. Furthermore, riboswitches that control the expression of key metabolic genes might serve as targets for antibacterial compounds, and the guanine riboswitches in some bacteria might be suitable targets for drug development. Aspects of the complex functions and the possible applications of purine riboswitches are outlined below.

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Figure 3 GuanineykkC tandem riboswitch system The secondary structure model for the guanineykkC tandem riboswitch arrangement from the purE mRNA of Moorella thermoacetica. The green-shaded regions indicate potential base-pairing interactions between the guanine riboswitch aptamer and the expression platform. Inset: predicted results of guanine and/or ykkC target ligand-binding to this riboswitch system. The letter G represents guanine and the question mark designates the ykkC target ligand. The larger font size indicates a higher concentration of that particular ligand in the cell.

The guanine aptamer and ykkC element can operate in tandem

An early report of a tandem riboswitch system described a riboswitch class that targets glycine (Mandal et al., 2004). This class commonly consists of two aptamers in tandem, residing upstream of an expression platform that usually controls genes involved in glycine processing in various bacterial species. Each aptamer is capable of recognizing a glycine molecule, and the two aptamers function together to exhibit co-operative ligand binding. This permits the riboswitch to bring about a greater change in gene expression in response to smaller changes in ligand concentration, which yields a more digital genetic element. A number of tandem riboswitch examples have been reported recently (Sudarsan et al., 2006). These examples occasionally carry distinct riboswitch classes that bind different ligands, but more commonly carry two riboswitches from the same class. In cases where the system consists of two separate riboswitch classes, there are two different ligands that can independently regulate gene expression (Sudarsan et al., 2006). In contrast, the presence of two members of the same riboswitch class yields an increase in the dynamic range for gene control and produces a more digital genetic response (Welz and Breaker, 2007). In several examples (Sudarsan et al., 2006), a guanine riboswitch aptamer resides in tandem with the candidate riboswitch RNA that is called ykkC (Barrick et al., 2004) in several bacterial species (Figure 3). Both elements are located upstream of the expression platform, which contains a transcription terminator stem. It is thought that in the absence of guanine, the guanine aptamer can prevent terminator-stem formation by an anti-terminator structure that sequesters nucleotides required for terminator formation. However, the anti-terminator cannot form in the presence of guanine, since some anti-terminator nucleotides are involved in forming the ligand-binding structure. Thus increased guanine concentrations are expected to repress downstream gene expression.

The ykkC RNA element typically resides upstream of an operon encoding a multidrug-resistance efux pump. Bioinformatics strategies were used to identify the motif as a riboswitch candidate (Barrick et al., 2004), but a ligand that binds to this motif has not yet been found. Nevertheless, ligand binding to the ykkC aptamer is predicted to deter formation of the terminator stem, because there appears to be an overlap between nucleotides required for terminator formation and the conserved sequence of the ykkC motif. It is possible that ligand binding either to the guanine aptamer or to the ykkC motif could supersede the effect of the other aptamer on gene control. Thus, the guanine and ykkC aptamers might function

Tandem riboswitches: Riboswitch architectures in which multiple riboswitches or their components are arranged in tandem to yield more complex gene control functions. Digital genetic element: A gene control element, such as a glycine riboswitch (Mandal et al., 2006), which produces a greater genetic response to increasing concentrations of the signalling compound than is possible with a simple 1-to-1 interaction between the compound and its sensor.

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Figure 4 Factors affecting kinetic and thermodynamic control of riboswitch systems A model depicting ve different variables (I through V) that contribute to determining whether a riboswitch is kinetically or thermodynamically controlled.

either independently or in combination to bring about opposing genetic responses, depending on concentrations of their target ligands in the cell (Figure 3, inset).
Riboswitches can function kinetically or thermodynamically

greater than the K d value. Thus the individual rate constant for ligand association is more important than the K d in inducing a riboswitch response. The adenine riboswitch in the 5 UTR of the B. subtilis pbuE gene appears to have the ability to be driven either kinetically or thermodynamically depending on various factors, such as the transcriptional time scale and the lifetime of the RNAligand complex (Wickiser et al., 2005; Gilbert et al., 2006b). The speed of the RNA polymerase, as well as transcriptional pausing at pause sites along the template must be taken into consideration, because any stalling by the RNA polymerase may allow the riboswitch system to reach equilibrium by the time the genetic decision must be made. In contrast, the adenine riboswitch upstream of the add gene in V. vulnicus is likely to be completely thermodynamically controlled (Rieder et al., 2007). This riboswitch regulates translation initiation, and adenine binding by this riboswitch can occur even after transcription of the expression platform, implying that the lifetime of transcription is not a factor in determining riboswitch character. Thus it is possible for different riboswitches within one class to operate differently under these two control regimes.
Guanine riboswitches are potential antibacterial targets

If a riboswitch controls gene expression by regulating transcription termination, then the genetic decision (gene activation or repression) will be controlled by either kinetic or thermodynamic parameters (or a combination of both) (Wickiser et al., 2005a, 2005b; Gilbert et al., 2006b; Rieder et al., 2007) (Figure 4). A riboswitch aptamer that reaches equilibrium before the genetic decision can be described as a thermodynamically driven riboswitch. In other words, such riboswitches should exhibit K d values that reect (with reasonable accuracy) the concentration of the metabolite needed to trigger riboswitch function in cells. However, if a riboswitch aptamer has insufcient time to reach binding equilibrium with its ligand, the concentration of ligand needed to trigger the riboswitch is dependent on the kinetics of the gene control process. For example, if the aptamer does not reach binding equilibrium by the time the RNA polymerase must make a genetic decision, then the speed of transcription dictates the concentration of metabolite needed to trigger the riboswitch. In this case, riboswitch-mediated gene regulation might occur only when the cellular ligand concentration is

Bacteria have been rapidly gaining resistance to currently used antibiotics (Neu, 1992; Gold and Moellering, 1996), which means that developing novel antibiotics for new targets is crucial for ghting these antibiotic-resistant bacteria. Riboswitches are currently being explored as possible antibacterial targets (Sudarsan et al., 2005; Blount and Breaker, 2006; Blount et al., 2006) due to their widespread use as gene-control elements in bacteria, as well as their ability to bind small molecules. The lysine analogues AEC (L-aminoethylcysteine) and D,L-4-oxalysine were found to be toxic to some bacterial species, at least in part by binding a lysine riboswitch and repressing gene expression (Sudarsan et al., 2003b; Blount et al., 2006). Also, the thiamine pyrophosphate analogue pyrithiamine pyrophosphate has been shown to inhibit bacterial growth by targeting thiamine pyrophosphate riboswitches (Winkler et al., 2002; Sudarsan et al., 2005). Analogues of different riboswitch ligands could potentially be designed that are targeted by riboswitches in vivo, which may prove

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sensing riboswitch classes described in the present review are quite small compared with some other riboswitch classes, and therefore might more easily emerge repeatedly as evolution progresses. Given the rapid pace of riboswitch discovery, it seems possible that riboswitches might be found that share the same secondary structure as the guanine, adenine and dG riboswitches, but recognize different purine-related molecules. Only a few key mutations are required within this general secondary structure to change the ligand specicities, but such core mutations also make it difcult for bioinformatics search strategies to identify them. In addition, there could be other purine-sensing riboswitches that have secondary structures and ligand-binding cores that are distinct from the guanine, adenine and dG riboswitches, but still use a riboswitch residue to create a canonical base-pair with the ligand. For example, the preQ1 riboswitch class probably recognizes preQ1 in this way, although its secondary structure differs from that of the other three purine-sensing riboswitch classes. Given the fundamental roles that purine-containing compounds must have played in ancient organisms, and the roles these compounds play in modern cells, it is likely that a greater diversity of purine-sensing aptamers might still be present in modern cells awaiting discovery and classication as unique motifs.

lethal to bacteria, particularly if the riboswitch in question controls essential genes. Guanine riboswitches might also be valid targets for new antimicrobial compounds. These riboswitches are found in many bacterial species, including some relevant pathogens such as Bacillus anthracis. The atomic resolution structure of the xpt motif bound to guanine has been determined (Batey et al., 2004; Serganov et al., 2004), which allows for the careful design of guanine analogues that can bind the aptamer. The riboswitch forms a tight binding pocket that surrounds the ligand almost completely, allowing for selective recognition of guanine and making it difcult for different compounds to bind to the aptamer. However, the 3D structure reveals unobstructed channels within the aptamerbinding pocket (Edwards et al., 2007), where additional chemical groups could potentially be added to create guanine analogues that will not disrupt ligand binding to the aptamer. Several guanine analogues have been synthesized that are bound by guanine riboswitches in vitro, and some of these analogues inhibit the growth of certain pathogenic bacteria (K. F. Blount, J. Lim, I. Puskarz, J. N. Kim, K. Link and R. R. Breaker, unpublished data). However, more experiments must be conducted to verify that guanine riboswitches can function as valid drug targets, particularly as it is not known whether the guanine analogues presently being examined have a lethal effect on bacteria due to the targeting of guanine riboswitches to the exclusion of other possible targets in cells. However, guanine riboswitches, and riboswitches in general, remain promising targets for future antibiotics.

Conclusions Given the widespread and highly conserved character of some riboswitch classes, the aptamer cores of these RNAs might be remnants of metabolite-sensing systems from the RNA World which presumably existed before the emergence of DNA and proteins (Joyce, 2002). Thus riboswitches (or the ancient version of riboswitches) would have been important in regulating metabolism in primitive life (Breaker, 2006). Perhaps the basic secondary structure of the guanine, adenine and dG riboswitches was present during the RNA World, and evolved into separate riboswitch classes over time. However, the size of all four purine-

Acknowledgements This work is supported by the Howard Hughes Medical Institute and by grants from National Institutes of Health (GM068819 and DK070270). J.N.K. is supported by the National Science Foundation Graduate Research Fellowship. R.R.B. is a cofounder of BioRelix, a company that has licensed intellectual property from Yale University regarding riboswitch technologies. References
*Articles of special interest Barrick, J.E., Corbino, K.A., Winkler, W.C., Nahvi, A., Mandal, M., Collins, J., Lee, M., Roth, A., Sudarsan, N., Jona, I., Wickiser, J.K. and Breaker, R.R. (2004) New RNA motifs suggest an expanded scope for riboswitches in bacterial genetic control. Proc. Natl. Acad. Sci. U.S.A. 101, 64216426 *Batey, R.T., Gilbert, S.D. and Montange, R.K. (2004) Structure of a natural guanine-responsive riboswitch complexed with the metabolite hypoxanthine. Nature 432, 411415 *Blount, K.F. and Breaker, R.R. (2006) Riboswitches as antibacterial drug targets. Nat. Biotechnol. 24, 15581564

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Purine sensing by riboswitches

Review
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Received 25 July 2007/31 August 2007; accepted 12 September 2007 Published on the Internet 17 December 2007, doi:10.1042/BC20070088

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