Metalloriboswitches: RNA-based inorganic ion sensors that regulate genes

Divalent ions fulfill essential cellular roles and are required for virulence by certain bacteria. Free intracellular Mg2+ can approach 5 mm, but at this level Mn2+, Ni2+, or Co2+ can be growth-inhibitory, and magnesium fluoride is toxic. To maintain ion homeostasis, many bacteria have evolved ion sensors embedded in the 5′-leader sequences of mRNAs encoding ion uptake or efflux channels. Here, we review current insights into these “metalloriboswitches,” emphasizing ion-specific binding by structured RNA aptamers and associated conformational changes in downstream signal sequences. This riboswitch-effector interplay produces a layer of gene regulatory feedback that has elicited interest as an antibacterial target.

Riboswitches are highly structured RNA motifs often located in the 5Ј-leader sequences of bacterial mRNAs (1)(2)(3). A riboswitch recognizes its cognate effector by a conserved aptamer domain, which elicits conformational changes in a downstream expression platform that alter transcription termination, translation initiation, message stability, or alternative splicing of the associated transcript (2,4,5). Because riboswitches evolved distinct molecular determinants to bind specific effectors, they have garnered interest not only for their elegant feedback mechanisms, but also for their potential to serve as novel drug targets. A prime example is the flavin mononucleotide (FMN) riboswitch, which can be inhibited by natural or synthetic ligand analogs that blunt the essential biosynthesis of riboflavin in bacteria (6 -10). At present, nearly 40 riboswitch classes that recognize Ͼ25 chemically distinct effectors have been described (11), providing many opportunities to interrupt key anabolic pathways. Conversely, a growing cohort of riboswitches activate genes involved in remediation of cellular toxins, such as S-adenosylhomocysteine (12), azaaromatics (13), guanidine (14), Mg 2ϩ (15), transition metals (16 -18), or magnesium fluoride (19). Here, we discuss the latter three classes of riboswitches that evolved elegant but distinct RNA folds to recognize specific divalent cations, leading to gene-regulatory changes that maintain cellular homeostasis. In some cases, metalloriboswitch affinity and cooperativity can rival that of proteins. Many excellent riboswitch reviews provide broad overviews of the field (2,3,11). As such, we focus here on the handful of known metalloriboswitches with an emphasis on their discovery, validation, specificity, and gene regulation, as well as their potential to serve as drug targets.

Mg 2؉ -I riboswitches are non-specific sensors that regulate Mg 2؉ import pumps
Mg 2ϩ is the most abundant intracellular, multivalent ion with free concentrations in bacteria ranging from 1 to 5 mM (20). Although numerous enzymes require Mg 2ϩ for catalysis, the ion can also fulfill structural roles by neutralizing charged DNA or RNA backbones or acting as a nexus for functional group coordination in RNA tertiary structure (21,22). The Escherichia coli 70S ribosome has ϳ170 coordinated Mg 2ϩ ions (23), and the lack of Mg 2ϩ in growth media leads to subunit dissolution and degradation (20). As a rule, bacteria employ a broad range of metallosensors and transporters to attain metal ion homeostasis (24). Some bacteria link Mg 2ϩ concentration to virulence genes to coordinate gene expression in cationdeficient host cells, such as macrophages (25). Salmonella enterica has three Mg 2ϩ importers: the widespread CorA channel and P-type ATPases MgtA and MgtB (26,27). Recent salmonellosis outbreaks caused by the Heidelberg serovar are alarming due to the multidrug resistance of this organism (28,29). In this and many other Gram-negative bacteria, mgtA expression is controlled by an upstream Mg 2ϩ -sensing riboswitch (known as the Mg 2ϩ -II class (11)) that regulates transcription via rho-dependent and RNase E degradation mechanisms (30 -32). An additional level of control appears to be exerted by a proline-rich open reading frame inside the mgtA riboswitch that works in concert with Mg 2ϩ -sensing to increase mgtA mRNA levels when Mg 2ϩ is low (33,34). Additional studies are needed to clarify the details of regulation, which appears to be present in only a handful of ␥-proteobacteria (11). A second class of Mg 2ϩ -sensing riboswitch known as the Mg 2ϩ -I (or M-box) is more broadly distributed in Gram-positive and some Gram-negative bacteria, where it is controls a variety of genes (15,25), including the widespread Mg 2ϩ -uptake channel mgtE (27). In Aeromonas hydrophila, mgtE mutations impair swarming and biofilm formation (35), which are important aspects of its pathogenicity. The defined Mg 2ϩ -I aptamer domain and readily discernible expression platform make it a model system for structure and function analysis, which provides a benchmark for additional comparisons herein.
The aptamer of the Mg 2ϩ -I riboswitch is not selective for Mg 2ϩ , unlike other metal-sensing riboswitches (see below), as shown by the variety of divalent ions that promote compaction of its fold and favor formation of a downstream intrinsic terminator hairpin within the expression platform (15,36). In this respect, Mg 2ϩ -I riboswitches likely function by sensing the most prevalent intracellular ion, Mg 2ϩ , which requires only modest affinity and selectivity, as corroborated by EC 50 values of 0.4 mM for Mg 2ϩ or Ca 2ϩ and 0.13 mM for Mn 2ϩ (36). Thus, Mg 2ϩ -I riboswitches promote homeostasis but are outperformed by regulatory metalloproteins in terms of selectivity (16). To investigate the metal-binding determinants, structures of the Mg 2ϩ -I riboswitch were determined in Mg 2ϩ -and Mn 2ϩ -bound states (15,36). The global architecture features three parallel helical domains comprising P1-P2-P6, P3-P4, and P5 (Fig. 1a). Tertiary interactions are stabilized by six Mg 2ϩ ions that promote compaction as shown biochemically (37). Four key Mg 2ϩ sites (I-IV) reside at the P1-P2-P5 interface, whereas sites V and VI are in the P4 internal loop. The former metal constellation (core 1) contributes to interhelical tertiary stability, whereas the latter (core 2) appears to orient the P4 loop to facilitate long-range contacts (36). Biochemical evidence also supports additional Mg 2ϩ binding, but these sites (core 3) were visible only in structures obtained from crystals grown from 5 mM Mn 2ϩ , which was used as a proxy for Mg 2ϩ . Importantly, only core 1 ions were present in all structures, including crystal forms harboring multiple Mg 2ϩ -I riboswitch copies per asymmetric unit. Moreover, sites I-III exhibited the highest phosphorothioate interference (37) with superimposable Mg 2ϩ and Mn 2ϩ coordination in pairwise structural com- The metal ions at each site coordinate in an octahedral manner. The site I Mg 2ϩ is coordinated by the non-bridging phosphate oxygens of G100, C102, and A103. A fourth ligand is contributed by the O4 keto moiety of U104 with two inner-shell water molecules completing the coordination sphere. Site II uses non-bridging oxygens from G100 and U23 with the remaining coordination sphere completed by waters. Site III uses two non-bridging oxygens from U24 and A25, as well as four inner-sphere waters. c, secondary structure depicting changes in conformational states that regulate transcription. Paired regions are colored as in a with backbone binding contributions to Mg 2ϩ from sites I-III depicted as green asterisks; additional Mg 2ϩ sites in P4 are depicted as green spheres. In the gene-off state, Mg 2ϩ binding at sites I-III promotes a conformation that destabilizes anti-terminator base pairing between the P1-P2 bulged loop (164 -172) and the 3Ј-tail (206 -213), favoring intrinsic-terminator hairpin formation that attenuates mgtE transcription, reducing Mg 2ϩ uptake by the MgtE channel. Conversely, low Mg 2ϩ favors RNA polymerase transcriptional read-through via an anti-terminator helix in place of P1. The full-length message leads to MgtE translation with increased Mg 2ϩ import into the cell. parisons (36). As such, it is worthwhile to describe these strong binding sites with a focus on how their tertiary contacts at these locations relate to gene regulatory conformations.
The site I Mg 2ϩ is coordinated by non-bridging phosphate oxygens at the transition of P5 into L5 with a fourth ligand contributed by the U104 base (Fig. 1b). Site II also uses nonbridging oxygens from P5 and P2 with the remaining coordination sphere completed by waters. Likewise, site III comprises two non-bridging oxygens from P2 and four waters. Shared coordination groups, such as the phosphate of G100, provide a plausible basis for cooperativity between Mg 2ϩ -binding sites (Hill coefficient 4.3) (36), which confers a sharp "digital" response to the effector over a narrow concentration range.
Gene regulation by the Mg 2ϩ -I riboswitch is predicated on Mg 2ϩ -mediated condensation of the RNA fold ( Fig. 1a) that favors sequestration of an anti-terminator sequence (164 -172) located between P1 and P2 (Fig. 1c). This effector-bound conformation promotes an alternate, intrinsic terminator harboring the 3Ј-anti-terminator sequence (206 -213), thus blocking transcription of the downstream mgtE gene, leading to attenuated Mg 2ϩ uptake by a diminished population of MgtE channels. Conversely, depleted cellular Mg 2ϩ alters the interface between P1-P2 and P5-L5 (sites I-III), promoting formation of an anti-terminator hairpin (Fig. 1c) that favors full-length mgtE transcription, resulting in Mg 2ϩ import. The presence of distinct Mg 2ϩ -sensing riboswitches in human pathogens suggests that these RNA elements could be targets for antibacterials, especially because the associated Mg 2ϩ transport genes are linked to virulence (discussed below).

Orphan riboswitch yybP-ykoY senses Mn 2؉ to activate intake pumps
Manganese is an essential trace element that plays key cellular roles, including facilitating the catalytic activity of various ribonucleotide reductases and superoxide dismutases expressed under low iron conditions or H 2 O 2 stress (38). Bacteria exhibit varied Mn 2ϩ requirements that reflect survival adaptations to specific microenvironments. E. coli can tolerate up to 20 mM Mn 2ϩ (39) but maintains cellular levels at ϳ1 M (24), whereas Bacillus subtilis has cytosolic levels of ϳ10 M (40). For some bacteria, Mn 2ϩ is toxic because in various enzymes it replaces Fe 2ϩ , which is not tolerated functionally (38). Conversely, Borrelia and Lactobacillus are members of small bacterial cohorts that use Mn 2ϩ with preference over iron (41,42), a strategy that bypasses host efforts to restrict iron levels, at least in the former case. Likewise, mutations in the macrophage-specific natural resistance-associated macrophage protein 1 (Nramp1) transporter-a Mn 2ϩ , Fe 2ϩ , and Zn 2ϩ uptake antiporter-led to diminished host resistance against Salmonella, Leishmania, and Mycobacterium (43), possibly resulting from a reduced capacity to remove divalent cations from bacteria-containing vacuoles in the host (44). Conversely, bacterial virulence can be attenuated by mutation of Nramp1 homologs of the pathogen, linking virulence to Mn 2ϩ uptake. Salmonella uses Mn 2ϩ transporters MntH (an Nramp1 homolog) and SitABCD (an ATPbinding cassette protein) (45), and a typhimurium serovar lacking both is avirulent (46). The mntH and sitABCD genes are repressed by the trans-acting Fur and MntR proteins in high Mn 2ϩ , although an additional cis-acting Mn 2ϩ -responsive riboswitch is hypothesized to reside in the 5Ј-leader of Salmonella mntH genes (47). This element requires further validation, but it ostensibly favors anti-terminator stem formation at low Mn 2ϩ concentrations, leading to MntH expression and Mn 2ϩ uptake. Exporters of Mn 2ϩ are also required by bacteria to attain homeostasis. The finding that the mntP (formerly yebN) encodes Mn 2ϩ efflux pumps, and is controlled by the Mn 2ϩ -responsive Fur and MntR proteins (48 -50), provided insight into a longstanding riboswitch mystery. The ubiquitous yybP-ykoY RNA motif, classified for years as an "orphan" riboswitch for lack of a known effector (51,52), was identified in the 5Ј-leaders of mntP genes, suggesting that Mn 2ϩ was the longsought ligand (17,18). Indeed, independent labs validated the yybP-ykoY or Mn 2ϩ riboswitch as an Mn 2ϩ sensor that controls expression of a P-type uptake pump (17,18). This provided a firm basis to understand how a metalloriboswitch discriminates between Mg 2ϩ and Mn 2ϩ , leading to cellular resistance against the toxic effects of a transition metal.
A structure of the Mn 2ϩ riboswitch revealed a four-way helical junction comprising tandem coaxially stacked helices (Fig.  2a). Mn 2ϩ -induced fold compaction based on chemical modification showed reduced loop flexibility at L1 and L3 and the base of P1 (17) with reactivity changes at U87 and A88 producing a K D, app value for Mn 2ϩ of 27 Ϯ 6 M. Chemical mapping also corroborated the structure, wherein two divalent ion-binding sites localize in bulged loops L1 and L3 to compose a prominent tertiary docking interface at the helical junction (Fig. 2, a  and b). At site I, Mn 2ϩ is coordinated by the N7 moiety of A41 and non-bridging phosphate oxygens from L3 and L1. The nearby site II pocket binds Mg 2ϩ using only oxygen atoms contributed by non-bridging groups of L1 and L3, as well as a single water molecule, consistent with the recalcitrance of Mg(H 2 O) 6 2ϩ to release its inner-sphere waters (53). Regarding the basis of Mn 2ϩ selectivity at site I, it is notable that Mn 2ϩ exhibits a more polarizable (softer) character in high-coordination-number environments, whereas Mg 2ϩ is oxophilic (harder) with less preference for nitrogen (53). Nonetheless, Mn 2ϩ functionally substitutes for Mg 2ϩ in many enzymes, and octahedral Mg 2ϩ will coordinate nitrogen (53). Consistent with these findings, riboswitch crystals prepared from 2.5 mM Mn 2ϩ incorporated Mn 2ϩ at both binding sites (17), but only site I was occupied when the Mn 2ϩ concentration was reduced to 100 M. Although biochemical cooperativity was not analyzed, interdependence of the ion-binding sites is likely based on shared coordination of non-bridging phosphate oxygens (Fig. 2b). As anticipated, 41A3 U and 41A3 G mutations are less responsive to Mn 2ϩ and are accompanied by enhanced L3 flexibility, consistent with rearrangement of the coordination pocket. It has been noted for proteins that insertion of a single nitrogen into a coordination sphere can exclude Mg 2ϩ and favor Mn 2ϩ (53). As such, it is reasonable that N7 of conserved A41 is a major selectivity determinant for Mn 2ϩ in the Mn 2ϩ riboswitch, which could account for the ability of Mn 2ϩ to bind even in the presence of relatively high Mg 2ϩ ion levels in the cytosol.
Gene regulation by Mn 2ϩ riboswitches occurs by two distinct mechanisms involving transcriptional and translational control (17,18). Structural analysis of the E. coli riboswitch showed that site I collapses in the absence of Mn 2ϩ , whereas site II remains intact and occupied by Mg 2ϩ (17). However, Mg 2ϩ alone did not stabilize the L1-L3 binding interface in chemical-modification experiments, which has implications for formation of mutually exclusive expression platform conformations that regulate transcription. At high Mn 2ϩ concentrations, the L1-L3 interface is well ordered (Fig. 2b), giving rise to a downstream transcription anti-terminator hairpin following P1 (Fig. 2c, top). This conformation favors polymerase readthrough, leading to full-length transcripts encoding the MntP (YoaB) efflux pump that confers Mn 2ϩ resistance. Conversely, the L3 sensor loop melts in the Mn 2ϩ -free state, favoring intrin-sic terminator formation (Fig. 2c, bottom) that leads to premature mntP termination. Similar conformational changes are envisioned for translational control, although the details of these changes are unclear at present.

NiCo riboswitches confer heavy metal resistance by sensing Ni 2؉ or Co 2؉
Heavy metals (i.e. Ͼ 5 g/cm 3 (39)) such as copper and silver have long been known to possess antimicrobial properties (54). Even essential metals at sufficiently high concentrations can be toxic, as is the case for trace nutrients Ni 2ϩ and Co 2ϩ . These ions can be incorporated into Fe-S proteins, leading to lethal effects that necessitate strict cellular control over their uptake  a). Mn 2ϩ is observed at site I and is preferred over Mg 2ϩ based on the N7 group of the A41 base. Non-bridging phosphate oxygens are contributed from U39, C40, U44, and C45 of L3, as well as G9 of L1. Site II is coordinated by non-bridging oxygens of G8, G9, and A10 of L1 and U39 and C45 of L3; an inner-sphere water molecule completes the coordination shell. Each non-bridging phosphate oxygen of G9 contributes to ion binding at sites I and II, providing a basis for cooperative binding. c, secondary structure diagrams depicting changes in Mn 2ϩ riboswitch conformational states that regulate transcription. Backbone contributions to Mn 2ϩ and Mg 2ϩ binding are depicted as magenta and green asterisks; Mn 2ϩ coordination at N7 of A41 is depicted as a magenta circle. In the gene-on state, Mn 2ϩ and Mg 2ϩ binding at sites I and II promote a conformation that facilitates base pairing of an anti-terminator hairpin above P1, favoring transcription of the mntP (yoaB) efflux pump that confers Mn 2ϩ resistance. Conversely, low Mn 2ϩ levels favor formation of an intrinsic terminator hairpin at the expense of the P1 helix and the L1-L3 ion-binding sites. A premature mntP (yoaB) message leads to reduced expression of the associated Mn 2ϩ efflux channel. (39,55). E. coli senses Ni 2ϩ and Co 2ϩ as repellants (56) and maintains intracellular levels in the low-to sub-M range (57). In minimal media, growth of E. coli is inhibited at 8 M Ni 2ϩ , and 160 M Co 2ϩ arrests S. enterica (58,59). Whereas Co 2ϩ is used for carbon rearrangements in the context of coenzyme B 12 , Ni 2ϩ is needed for enzymes such as hydrogenase and urease (24). Once inside the periplasm, ABC transporters and NiCo permeases are the major modes of bringing Ni 2ϩ and Co 2ϩ into the bacterial cell (24). The non-specific CorA transporter also facilitates cytosolic accumulation (27). To attain homeostasis, bacteria have cadmium-zinc-cobalt (czc) genes that encode efflux pumps and cation diffusion facilitators such as CzcD. Like the Mg 2ϩ -I and Mn 2ϩ riboswitches, comparative sequence analysis revealed a structured RNA motif upstream of czcD genes in multiple bacterial species (16). Isolated czc RNA motifs showed in vitro K D , app values of 5.6 and 12 M for Co 2ϩ and Ni 2ϩ , with five metal-responsive regions in the four-way helical junction core. Comparatively weaker binding (K D , app of 220 M) was seen for Mn 2ϩ .
The czc motif or "NiCo" riboswitch must selectivity bind Co 2ϩ or Ni 2ϩ amid much higher intracellular Mg 2ϩ concentrations. To elucidate the basis of affinity and recognition of these ions, a riboswitch structure was determined in the presence of Co 2ϩ (16). Like the Mg 2ϩ -I and Mn 2ϩ riboswitches, the NiCo structure lacks pseudoknot interactions and exhibits close packing, whereby four helices coaxially stack in pairs (Fig. 3a). Three co-planar Co 2ϩ -binding sites (I-III) reside at the interface between L2-3 and L4-1 with a fourth site (IV) at the base of P2. Like the Mn 2ϩ riboswitch motif, Co 2ϩ or Ni 2ϩ selectivity is conferred by the purine N7 moiety, with sites I-III each coordinating two imines (Fig. 3b). Hill coefficients of 2.0 and 1.6 were measured for Co 2ϩ and Ni 2ϩ , using the Clostridium botulinum NiCo riboswitch. The structure nicely explains such positive cooperativity in that sites I and II coordinate the N7 and 2Ј-OH groups from opposing L4-1 and L2-3 nucleotides that knit the junction together. In this manner, the G46 N7 group coordinates site II, and its 2Ј-OH coordinates site I; conversely, the N7 group of G87 coordinates site I, and its 2Ј-OH coordinates sites II (Fig. 3b). As such, Co 2ϩ binding at one site stabilizes coordination at the adjoining site. N7-deaza substitutions at G87 and G88 underscore the importance of N7 coordination. The former mutation cannot support Co 2ϩ -dependent stabilization at G46, indicating that sites I and II are indeed linked. In contrast, N7-deaza G87 did not affect site III, consistent with its distal location from site I. At present, site IV appears to provide stabilization of P1-P2, with the site exhibiting a significantly lower anomalous signal than sites I-III (16).
Inspection of the NiCo riboswitch expression platform suggests the formation of two mutually exclusive conformations. At low Co 2ϩ or Ni 2ϩ concentrations, an intrinsic terminator hairpin is favored, whereas elevated ion levels support polymerase read-through leading to czcD expression (Fig. 3c). This mechanism was confirmed by in vitro transcription assays, which yielded full-length products only in the presence of cognate effector ions. Regulation was validated in vivo when a representative czc motif from Clostridium scindens was placed upstream of a putative cation efflux gene. Whereas levels of the associated mRNA increased with increasing Ni 2ϩ , a similar analysis using non-cognate ions did not affect downstream transcript levels (16). Overall, the cooperative binding and unique mode of metal recognition by the NiCo riboswitch impart high selectivity, enabling feedback to minute changes in Co 2ϩ or Ni 2ϩ in a milieu of competing cellular ions to confer heavy-metal resistance. Notably, NiCo riboswitches are present in Listeria monocytogenes, a common foodborne pathogen (60) that is of concern because of the identification of multi-antibiotic resistance isolates in "ready-to-eat"' foods (61).

Fluoride riboswitches require Mg 2؉ to sense and regulate export of a cellular toxin
Fluorine is the most electronegative and reactive element and is found predominantly in the biosphere as the fluoride ion (F Ϫ ). Although a trace element, fluorine is ubiquitous with levels ranging from 1.2 ppm (mg/liter) in seawater to 200 ppm in some soils (62,63). High levels of F Ϫ are toxic to bacteria, fungi, and animals (62,64,65), and in many regions F Ϫ is added at 0.7 ppm to municipal water supplies as an anticaries agent (66). The beneficial and toxic effects of F Ϫ arise from similarities in its charge and radius to hydroxide (67). The distinct antimicrobial properties of F Ϫ are derived from its affinity for divalent cations. F Ϫ is toxic to enolase because it stabilizes a bis-MgF-PO 4 complex that mimics the reaction intermediate (68 -70).

MgF 3
Ϫ and AlF 3 complexes also form trigonal-bipyramidal mimics of phosphoryl-transfer transition states (71) to inhibit catalysis by this essential class of enzymes.
Chronic contact with F Ϫ contributed to the evolution of fluoride toxicity-resistance factors that stimulate expression of genes to ameliorate intracellular F Ϫ levels, including transporters (19,(72)(73)(74). The observation that many bacteria and archaea use fluoride-responsive RNAs to regulate such genes suggests that F Ϫ -sensing riboswitches arose as part of an ancient strategy to achieve F Ϫ resistance (19,75). Such riboswitches were first identified as conserved, non-coding motifs upstream of genes such as crcB, which encodes a fluoridespecific channel (19,72,73), as well as metabolic enzymes such as enolase (19,76). Biochemical experiments on the ϳ80-nucleotide crcB 5Ј-leader RNA confirmed a 1:1 binding stoichiometry for F Ϫ only in the presence of Mg 2ϩ (K D, app ϳ60 -135 M) (19,77), with no apparent binding to Cl Ϫ , Br Ϫ , I Ϫ , or the infused gases CO and NO. Mutations that alter conserved core sequences or secondary structures of the riboswitch adversely impact F Ϫ binding, as expected for molecular determinants that evolved to bind a specific effector (78). Fusion of the Bacillus cereus 5Ј-leader of crcB to a lacZ reporter in B. subtilis revealed F Ϫ -dependent ␤-galactosidase activity consistent with its modulation of an intrinsic transcription terminator. Evidence for translational control by an alternative expression platform came from a crcB riboswitch from Pseudomonas syringae that lies upstream of eriC genes. This riboswitch was used to control lacZ in an E. coli crcB knock-out strain that is ϳ200-fold more sensitive to F Ϫ than WT (19). This work demonstrated a role for CrcB in reducing cellular F Ϫ levels and provided insight into eriC function and selectivity. Specifically, EriC F proteins rescued growth of E. coli crcB knock-out cells under high F Ϫ conditions, suggesting that these genes are func-tionally homologous (19). Accordingly, it is now established that EriC F proteins are fluoride-selective antiporters (19,74).
To elucidate the mode of F Ϫ sensing and the underlying basis for gene regulation, the structure of a fluoride riboswitch was determined (77). The structure confirmed that the conserved aptamer folds as an HL out pseudoknot (Fig. 4a) (79). Unexpectedly, a constellation of five backbone phosphates at the topological confluence of P2, L1, and L3 chelate three Mg 2ϩ ions crucial for F Ϫ sensing. These cations form the vertices of a nearly equilateral triangle with a central cavity that is ideal for F Ϫ coordination (radius 1.30 Å) (Fig. 4b), while selectively excluding larger halides such as Cl Ϫ (radius 1.81 Å) (80). Similar tri-Mg 2ϩ coordination of F Ϫ has been observed previously in proteins (81,82).
The gene regulatory functions of Thermotoga petrophila and B. cereus fluoride riboswitches likely entail modulation of an intrinsic terminator that forms at low F Ϫ concentration (Fig.  4c), ostensibly reducing CrcB levels. NMR data corroborate F Ϫ -dependent compaction of the T. petrophila crcB motif with interconversion of the effector free state into a compact form  a). Sites I-III interact directly with bases G47 and G87, G46 and G88, and A14 and G45, which are conserved among most czc homologs. G46 and G87 each contribute to coordination at sites I and II via purine N7 and ribose hydroxyl groups that form the basis of cooperativity during Co 2ϩ or Ni 2ϩ binding. In contrast to the Mg 2ϩ -I and Mn 2ϩ riboswitches that make use of non-bridging phosphate oxygens to coordinate Mg 2ϩ or Mn 2ϩ , NiCo riboswitch coordination utilizes one or two N7 moieties, 2Ј-OH groups, and water molecules to bind Co 2ϩ and presumably Ni 2ϩ . c, secondary-structure diagram depicting effector-dependent conformational changes that regulate transcription; asterisks indicate sites of purine N7 Co 2ϩ coordination in b, and pentagons show coordination from ribose 2Ј-OH groups. Transcriptional regulation by the NiCo riboswitch parallels that of the Mn 2ϩ riboswitch because Co 2ϩ or Ni 2ϩ sensing favors an RNA fold that allows RNA polymerase to read through the transcript, leading to a full-length message that confers heavy-atom resistance when translated. In the effector-free state, P4 remodels to form a terminator hairpin leading to premature transcription termination, potentially resulting in Co 2ϩ or Ni 2ϩ accumulation in the cell. (77). Mapping the Mg 2ϩ coordination sites onto the riboswitch structure suggests that structurally disparate regions in L1, P2, and L3 may be pre-organized early in transcription to coordinate F Ϫ and Mg 2ϩ , which promotes complete folding of P2 before the 5Ј-end of the terminator helix folds into a hairpin that arrests transcription. An alternative expression platform has been identified in the F Ϫ riboswitch from P. syringae in which a Shine-Dalgarno sequence in the 3Ј-tail is sequestered from the 16S rRNA of the ribosome under low F Ϫ levels (19), thus attenuating translation initiation required for EriC F synthesis.

Prospects for therapeutic targeting and discovery of new metalloriboswitches
A handful of riboswitches are known to bind metabolite analogs that alter gene expression and bacterial growth (6 -9, 83, 84). Although metalloriboswitches do not bind organic ligands, they are expected to be susceptible to small molecules that target conserved binding pockets (85). Such small molecules will likely hit other members of the same class, and any escape mutant will impede normal ion binding and gene regulation. High-throughput and fragment-based screening, as well as structure-guided methods, have yielded compounds that target metabolite-sensing riboswitches (6 -9, 83, 86, 87), and these approaches will likely succeed for metalloriboswitches. Small molecules that lock the Mg 2ϩ -I riboswitch into a compact gene-off state (e.g. Fig. 1a) would attenuate MgtE expression, depriving bacteria of Mg 2ϩ . Deletion of mgtE in B. subtilis yielded a defect requiring Ͼ25 mM Mg 2ϩ for growth; by contrast, mgtA deletion-controlled by some Mg 2ϩ -II riboswitches-had no effect (88).
As for the Mn 2ϩ and NiCo riboswitches, these regulators are important when iron is scarce or H 2 O 2 is present, such as inside a host cell. Therein, iron enzymes of the pathogen can lose activity, and Mn 2ϩ import by MntH becomes essential (45,89). Stabilizing the mntH riboswitch in a gene-off state could reduce virulence, although the details of this conformation are unclear. The riboswitch folds as an HL out pseudoknot wherein F Ϫ ion (pink) sensing occurs by three Mg 2ϩ ions (green) coordinated between loops L1 and L3 that mediate long-range contacts between coaxially stacked helices P1 and P2; loop L2 ϭ 0. b, close-up view of the ion-sensing pocket (boxed in a), revealing a trigonal constellation of octahedrally coordinated Mg 2ϩ ions, I-III, which share a central F Ϫ ion. Like the Mg 2ϩ -I and Mn 2ϩ riboswitch, non-bridging phosphate oxygens provide coordination sites for Mg 2ϩ with inner-sphere water molecules completing the coordination sphere. c, secondary-structure diagram depicting effector-dependent conformational changes that regulate transcription; asterisks indicate the sites of backbone oxygen coordination to Mg 2ϩ in b. In the presence of fluoride, the 5Ј-end of a terminator hairpin is buried by the pseudoknot, leading to transcription of a full-length message and translation of a CrcB channel that exports F Ϫ to confer resistance. In the absence of fluoride, there is no need for F Ϫ export, and the intrinsic terminator hairpin is favored, leading to the gene being maintained in an off state.
Similarly, small molecules that fix the mntP riboswitch in a gene-on state (Fig. 2a) would also lower Mn 2ϩ levels. Importantly, heavy metals can enter bacteria by non-specific import that requires regulated export for homeostasis (90). Whereas the czcD gene confers Co 2ϩ and Ni 2ϩ resistance, its deletion in Streptococcus pneumoniae led to increased Zn 2ϩ sensitivity (IC 50 of ϳ250 M) (91). Zn 2ϩ varies in human tissues from 20 M in serum to 230 M in lung (92), and high levels can impair iron enzymes by mismetallation (93). It is conceivable that trapping the NiCo riboswitch in a gene-off state (Fig. 3c) would impair growth under certain environmental conditions (91).
Deletion of crcB increases bacterial sensitivity to F Ϫ (19), but certain compounds are also known to enhance F Ϫ sensitivity (94 -96). This work supports the prospect of targeting F Ϫ riboswitches in concert with supplemental F Ϫ (75). Such molecules would be selected to promote gene-off states (Fig. 4c), blocking F Ϫ export. Beyond bacteria, CrcB homologs confer F Ϫ resistance in fungi and yeast (73) but are absent in mammals. In general, F Ϫ riboswitches are missing in eukaryotes (11), supporting prospects for antibacterial development. Overall, the metalloriboswitch field would benefit greatly by placing greater emphasis on analysis in the context of infectious disease models.
The abundance of representatives for various riboswitch classes obeys a power-law relationship that suggests ϳ1000 new classes remain to be discovered (11). In terms of new metalloriboswitches, it is reasonable to consider that magnesium, calcium, manganese, iron, cobalt, copper, and zinc are essential for most life forms, whereas vanadium, nickel, and tungsten are present only in bacteria (97). Each metal represents a likely effector, but it is reasonable to expect new riboswitches will sense the known ions described here. Such aptamers might possess unique sequences or use known scaffolds with variations to alter specificity (98). For example, the Mg 2ϩ -I riboswitch might be co-opted to bind softer metals by replacing its oxygen ligands with nitrogen. Conversely, adding an equatorial oxygen ligand could favor pentagonal bipyramidal coordination of Ca 2ϩ , which is maintained at 100 -300 nM in bacteria (99). Perhaps the most likely effector is iron because it is the fourth most abundant element in the earth's crust and is essential for most life. A precedent exists for small RNA regulation of iron-storage proteins in bacteria (100). Overall, the discovery of new and possibly rare metalloriboswitches will require traditional bioinformatic methods along with targeted searches (30). All evidence suggests that the number of metal-sensing RNAs will continue to grow (11) and that this niche of genetic control is worth further consideration on the path to restoring a waning antimicrobial armamentarium.