Bacterial Riboswitches Cooperatively Bind Ni2+ or Co2+ Ions and Control Expression of Heavy Metal Transporters

Article

Bacterial Riboswitches Cooperatively Bind Ni2+ or Co2+ Ions and Control Expression of Heavy Metal Transporters Graphical Abstract

Authors Kazuhiro Furukawa, Arati Ramesh, ..., Wade C. Winkler, Ronald R. Breaker

Correspondence [email protected] (W.C.W.), [email protected] (R.R.B.)

In Brief Metal ions serve many essential roles but are toxic in excess; therefore, organisms utilize proteins that allosterically couple metal-sensing sites to control of gene expression. Furukowa et al. discover that certain noncoding RNAs can also perform this task, suggesting that such metalloregulation is not limited to proteins.

Highlights d

NiCo RNA is the first conserved riboswitch class that responds to transition metals

d

NiCo selectively recognizes cobalt or nickel, binding with positive cooperativity

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NiCo riboswitches control metal homeostasis by regulating metal transport proteins

Furukawa et al., 2015, Molecular Cell 57, 1088–1098 March 19, 2015 ª2015 Elsevier Inc. http://dx.doi.org/10.1016/j.molcel.2015.02.009

Accession Numbers 4RUM

Molecular Cell

Article Bacterial Riboswitches Cooperatively Bind Ni2+ or Co2+ Ions and Control Expression of Heavy Metal Transporters Kazuhiro Furukawa,1,5,7 Arati Ramesh,4,6,7 Zhiyuan Zhou,1,7 Zasha Weinberg,2 Tenaya Vallery,3 Wade C. Winkler,4,* and Ronald R. Breaker1,2,3,* 1Department

of Molecular, Cellular and Developmental Biology, Yale University, New Haven, CT 06520, USA Hughes Medical Institute, New Haven, CT 06520, USA 3Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT 06520, USA 4Department of Cell Biology and Molecular Genetics, University of Maryland, College Park, MD 20742, USA 5Present address: Faculty of Pharmaceutical Sciences, The University of Tokushima, 1-78-1 Shomachi, Tokushima 770-8505, Japan 6Present address: National Centre for Biological Sciences, GKVK Campus, Bellary Road, Bangalore 560065, India 7Co-first author *Correspondence: [email protected] (W.C.W.), [email protected] (R.R.B.) http://dx.doi.org/10.1016/j.molcel.2015.02.009 2Howard

SUMMARY

Bacteria regularly encounter widely varying metal concentrations in their surrounding environment. As metals become depleted or, conversely, accrue to toxicity, microbes will activate cellular responses that act to maintain metal homeostasis. A suite of metal-sensing regulatory (‘‘metalloregulatory’’) proteins orchestrate these responses by allosterically coupling the selective binding of target metals to the activity of DNA-binding domains. However, we report here the discovery, validation, and structural details of a widespread class of riboswitch RNAs, whose members selectively and tightly bind the low-abundance transition metals, Ni2+ and Co2+. These riboswitches bind metal cooperatively, and with affinities in the low micromolar range. The structure of a Co2+-bound RNA reveals a network of molecular contacts that explains how it achieves cooperative binding between adjacent sites. These findings reveal that bacteria have evolved to utilize highly selective metalloregulatory riboswitches, in addition to metalloregulatory proteins, for detecting and responding to toxic levels of heavy metals.

INTRODUCTION At high concentrations, heavy metal ions, such as Co2+, Ni2+, Zn2+, and especially Cu2+, can form undesirable complexes with various metabolites, and biopolymers, which have toxic effects on cells (Agranoff and Krishna, 1998; Nies, 1999, 2003). Yet, metals are often essential as enzyme cofactors or as structural components of cellular metabolites and proteins. A balance in the cellular metal concentration is thus key to bacterial lifestyles. To achieve heavy metal ion homeostasis, bacteria use a combination of chelators, chaperones and metal-utilizing en-

zymes (Waldron and Robinson, 2009), as well as several classes of selective metal ion transporters such as cation diffusion facilitator (CDF) proteins, P type export ATPase proteins, and resistance nodulation division proteins (Nies, 2003). Tight regulation of the expression and function of these different metal ion homeostasis proteins is required to maintain ion concentration within their optimal ranges. A variety of metal-sensing regulatory (‘‘metalloregulatory’’) proteins have been discovered for these purposes. Indeed, at least ten diverse structural families of metal sensors have been identified that regulate gene expression in response to either toxicity or deprivation of target metals (Ma et al., 2009). These metal sensor proteins bind one or several cognate metal ions and exclude all other metal ions, in part by exquisite tuning of the binding pockets to coordination chemistry of the target ions. Binding of only the correct metal ion then triggers changes in the protein structure, and dynamics such that the transcription factors are either activated or de-activated for DNA-binding proficiency. However, it is still unknown whether or not metalloregulation is strictly limited to these classes of protein-based sensors. In recent years, many different genetic regulatory roles have been discovered for signal-sensing, noncoding RNAs, including many functions previously attributed only to proteins. It is therefore possible that direct metalloregulation is yet another underappreciated function for noncoding RNAs, especially given the fact that RNA polymers also possess functional groups capable of coordinating metal ions. In general support of this hypothesis, several rare examples of ion-responsive riboswitches have been discovered in the past few years that respond to Mg2+, F
, or Mn2+ (Baker et al., 2012; Cromie et al., 2006; Dann et al., 2007; Shi et al., 2014). Riboswitches are RNAs that selectively bind small metabolites and control the expression of genes whose protein products function in biochemical pathways related to their ligands (Breaker, 2011; Mandal and Breaker, 2004). Previously, two distinct Mg2+-responsive riboswitches (FerreD’Amare and Winkler, 2011) were discovered for genetic regulation of Mg2+transport proteins in Bacillus subtilis (Dann et al., 2007; Ramesh and Winkler, 2010) and Salmonella species (Cromie et al., 2006). Structural and biophysical characterization of

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Figure 1. Structure and Function of czc Motif RNAs (A) Consensus sequence and structural model for czc motif RNAs. Nucleotides present in greater than 97%, 90%, and 75% of the representatives are depicted as red, black and gray, respectively. Predicted base-paired substructures (P1 through P4), covarying nucleotides (green shading), and non-covarying nucleotides (red shading) are shown. (B) RNA motif 95 Cbo from Clostridium botulinum. 50 G residues (lowercase) were added to facilitate in vitro RNA transcription. Disruptive (M1) and restorative (M2) mutations are depicted. Sites of ion-induced changes in spontaneous cleavage are derived from (C) and Figures 2A and S1A. (C) In-line probing analysis of the 95 Cbo RNA with five divalent metal ions. 50 32P-labeled precursor RNAs were subjected to in-line probing without ligand (
) or with 0.3 mM of the divalent ion denoted for each lane. Non-reacted (lane NR) partial digestion of RNA with RNase T1 for cleavage after G residues (lane T1) and alkaline conditions for cleavage at every position (lane OH) are shown. Red brackets highlight cleavage products whose yields are altered by ligand addition. (D) Periodic table highlighting different effects of metal ions on the czc RNA. The characteristics of various metal cations (20 mM) were established by in-line probing shown in Figure S1.

the B. subtilis Mg2+-responsive riboswitch revealed that accumulating Mg2+ ions trigger dramatic compaction of the signalsensing domain (aptamer). This compacted structure consists of three, approximately parallel helical columns that are stabilized by an array of tertiary interactions (Dann et al., 2007; Ramesh et al., 2011) This structure sequesters an oligonucleotide sequence normally required for an anti-terminator helix, thereby coupling divalent metal ion concentrations to the default formation of an intrinsic terminator, resulting in cessation of transcription. However, while the Mg2+-responsive riboswitch utilizes individual divalent ion-binding pockets, these metal-binding sites are not selective, and the metal-chelated compact configuration can be induced in vitro by many different metal cations. In fact, the riboswitch is only responsive to Mg2+ in vivo because Mg2+ is the only divalent metal cation to reach intracellular concentrations high enough to trigger the necessary compacted configuration. Therefore, while the Mg2+-responsive riboswitch is important for maintaining Mg2+ homeostasis, it does not exhibit performance characteristics that rival that of metalloregulatory proteins, particularly with respect to metal selectivity. However, in this work, we identify the first conserved RNA class that selectively responds to low-abundance metal ions, Co2+ and Ni2+. These ‘‘NiCo’’ riboswitches bind their target metal ligands despite much higher concentrations of other metal cations, indicating that metal sites in NiCo are evolutionarily fine-

tuned to the coordination chemistry of Co2+ and Ni2+ ions. Positive cooperativity between metal sites in the NiCo aptamer suggest that the genes controlled by these riboswitches are likely to be induced by even small changes in the cellular concentrations of these toxic heavy metals. We have resolved an atomic resolution model of Co2+-bound NiCo RNA using X-ray crystallography. The structure reveals the nature and geometry of multiple interconnected Co2+ sites, thus elucidating the structural framework for metalloregulation by a riboswitch RNA. Moreover, we demonstrate that a Clostridium scindens NiCo riboswitch regulates the expression of Co2+ transporters in vivo, most likely for preventing accumulation of intracellular cobalt. Together, our findings discover a metalloregulatory riboswitch RNA that is widespread in bacteria and is functionally equivalent to metalloregulatory proteins for control of transition metal homeostasis. RESULTS Discovery of a Conserved RNA Element Associated with Metal Transporters Using comparative sequence analysis to search bacterial genomes from the order Clostridiales and metagenomic sequences, we discovered a novel structured RNA class (Figure 1A; Supplemental Information) consisting of four base-paired regions (P1 through P4). The paired regions show little sequence

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Figure 2. Cooperative Ligand Binding of a czc Motif RNA

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(A) In-line probing analysis of the 95 Cbo RNA with increasing concentrations of Co2+ or Ni2+ (0.78 mM to 200 mM) shows five regions of structural changes in the RNA (red brackets numbered 1 to 5). Lanes are marked as in Figure 1. (B) Plot of the normalized fraction of RNA cleavage versus Co2+ derived from the data in (A) shows half-maximal structural change at 6.5 mM Co2+. (C) Hill plot of the binding data observed for Co2+, Ni2+, and Mn2+. Estimated Hill coefficients (n) are derived from the slopes of the lines. R2 values for the curve analyses are 0.995, 0.995, and 0.998 for Co2+, Ni2+, and Mn2+, respectively. Also see Figure S2 for Ni2+ and Mn2+ binding assays.

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conservation but a high degree of covariance, suggesting that the base pairing potential of these stems is evolutionarily maintained and likely important. The junction of the paired helices is composed of a highly conserved nucleotide core. Nearly half of the 569 ‘‘czc motif’’ RNA representatives identified reside upstream of czcD genes, which encode for a subfamily of CDF proteins involved in the efflux of cobalt, zinc, and cadmium (czc) in organisms from all three domains of life (Anton et al., 1999; Bouzat and Hoostal, 2013; Grosse et al., 2004). Thus, we speculated that czc RNAs likely function as riboswitches for specific heavy metal ions. Selective Binding of Co2+ and Ni2+ Ions to the Conserved RNA To test if the czc motif responds to metal ions, we used in-line probing (Regulski and Breaker, 2008; Soukup and Breaker, 1999), which reveals ligand-induced changes in secondary and tertiary structure. Using a 50 32P-labeled 95-nt czc motif RNA from Clostridium botulinum (termed 95 Cbo, Figure 1B), we initially assessed the effects of five different divalent metal ions (Co2+, Ni2+, Cu2+, Zn2+, and Cd2+) on the pattern of spontaneous RNA cleavage. Only two of these cations, Co2+ and Ni2+, strongly modulate RNA structure in all five regions (Figures 1C and 2A). Nucleotides in these metal-responsive regions cluster in or near the core of the four-stem junction (Figure 1B), indicating the conserved portions of this RNA motif form one or more specific binding pockets for Co2+ and Ni2+. The strong natural conservation of all four predicted stems suggests that these substructures are also important for aptamer function. Indeed, mutation M1 that disrupts base pairing within stem P2 eliminates the majority of Co2+ or Ni2+ binding at cation concentrations as high as 100 mM, whereas an additional compensatory mutation M2, which restores base pairing, also restores metal binding (Figures 1B and S1A). A more complete binding survey conducted using various cations at 20 mM (which is higher than the apparent dissociation

constant [KD] for Co2+ and Ni2+ as calculated below), produced five general outcomes (Figures 1D and S1B). A metal ion could (i) bind tightly, (ii) bind weakly, (iii) not bind, (iv) disrupt RNA structure formation or alter gel mobility of the RNA, or (v) cause rapid RNA degradation. Only Co2+ and Ni2+ appear to bind tightly, although inclusion of Mn2+ generates a pattern of spontaneous cleavage products that are similar to that for Co2+ and Ni2+, despite its substantially poorer affinity (see below). Some cations (Be2+, Sc3+, Cr3+, Ru3+, and Rh3+) caused anomalous mobility during gel electrophoresis. Similar adverse effects were observed previously for some of these same metal cations when tested with a self-cleaving ribozyme (Zivarts et al., 2005). These apparent nonspecific interactions between certain metal ions and RNA in general are unlikely to be relevant to the biological function of czc motif RNAs. Five of the metals tested (La3+, Os3+, Hg2+, Pb2+, and Sn2+) promote the spontaneous degradation of RNA. It has been shown previously that Pb2+ generally catalyzes the rapid cleavage of RNA, likely by deprotonating 20 -hydroxyl groups (Ciesio1ka et al., 1998), which increases the nucleophilicity of the 20 oxygen and promotes cleavage of adjacent phosphodiester bonds (Li and Breaker, 1999). Thus, our assays cannot be used to determine whether these latter cations are selectively bound by czc RNAs in a manner similar to Co2+ and Ni2+. In-line probing was used to establish the binding affinities of 95 Cbo RNA for Co2+, Ni2+, and Mn2+ (Figures 2A and S2). Co2+ exhibits an apparent dissociation constant (KD) of approximately 6.5 mM, but the binding data are best represented by a curve wherein the Hill coefficient is greater than 1 (Figures 2B and 2C). Similar results are observed for Ni2+ (Figures 2C and S2B), revealing that binding is not a simple one-to-one interaction between RNA and Co2+ or Ni2+, but rather involves the cooperative binding of more than one cation by each aptamer. In contrast, Mn2+ exhibits weaker binding and no evidence for cooperative binding (Figures 2C and S2A–S2C). Some riboswitches (Trausch et al., 2011) are known to cooperatively bind two identical ligands, although the vast majority of riboswitch classes exhibit simple one-to-one complex formation as demonstrated by numerous atomic-resolution riboswitch

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structures (Garst et al., 2011; Roth and Breaker, 2009; Serganov and Patel, 2012). Structural Resolution of Co2+-Bound NiCo RNA To gain deeper mechanistic insight and to elucidate the molecular architecture of the czc motif RNA (henceforth referred to as the NiCo riboswitch), we used X-ray crystallography. NiCo RNA aptamers from many organisms were produced in milligram quantities in vitro and screened for crystal growth in the presence of various divalent cations. Crystals most useful for X-ray diffraction were obtained for the Erysipelotrichaceae bacterium representative in the presence of Co2+. These crystals yielded a structural model of the Co2+-bound aptamer at a resolution of 2.6 A˚ and with an Rwork and Rfree of 20% and 23.5%, respectively (Figure 3A; Table 1). In-line probing of the NiCo aptamer from E. bacterium (termed 97 Eba) confirmed that selectivity for Ni2+ and Co2+ is conserved across this RNA class (Figure S3). The 97 Eba RNA displays selective, tight, and cooperative binding for Ni2+ and Co2+ with apparent binding affinities of 5.6 mM for Co2+ and 12 mM for Ni2+. The global architecture of the NiCo RNA is remarkably different from other known riboswitch structures. Unlike most riboswitches, NiCo adopts a structure that lacks both long-range tertiary interactions and close helical packing. Instead, the RNA adopts a twisted, ‘‘H’’-like conformation (Figures 3A and 3B), where two sets of coaxially stacked helices (P1 with P2, P3 with P4) are arranged at a 30 angle (Figures 3D–3F). Electron density corresponding to two nucleotides of the L4 loop could not be visualized clearly and are thus not included in the model. However, the L4 loop is the least conserved region of NiCo riboswitches (Supplemental Information), does not undergo ligandmediated structural changes (Figure 1B), and therefore is not likely to be of functional importance. Four Co2+ ions were located experimentally using Co2+ anomalous dispersion data (Figure 4E). Three Co2+ sites C1, C2, and C3 reside in the four-stem junction, lying in close proximity on a plane perpendicular to the long axis of the aptamer (Figures 3B–3F). Nucleotides G45, G46, and G47 from junction J2/3 and nucleotides G87 and G88 from junction J4/1 directly coordinate Co2+ ions 1, 2, and 3 (Figure 4), which together appear to stabilize the junction. Cobalt-coordinating nucleotides additionally interact with junction nucleotides from the base of the P1 and P3 helix (G47:C60, G88:C16, G45:A17, and A14:A89) to orient the flanking helices and possibly stabilize the RNA structure. Sequence analysis of NiCo RNAs suggests that nucleotides between 79 through 89 could base pair to form the P4 stem or alternately base pair with downstream nucleotides to form the left shoulder of the intrinsic terminator stem. In the NiCo structure, these nucleotides directly contact Co2+ ions. This supports our hypothetical mechanism for gene control wherein metal binding stabilizes an antiterminator structure in the NiCo aptamer. Also, the nucleotides that undergo robust structural changes (Figure S3) cluster near the Co2+ binding sites (Figure 3C), supporting a relationship between binding of the metals and the structural changes observed by in-line probing. Few ligand-mediated structural changes occur outside of this region (Figure S2), which agrees well with the absence of long-range tertiary contacts revealed by our model. Size-exclusion chroma-

tography of NiCo shows minimal changes in its global conformation in the presence of Mg2+ or Co2+, thus suggesting that metal sites in NiCo are likely to be preformed (Figure S5A). Binding sites for two Mg2+ and seven K+ ions were also identified (Figures 3D–3F). The positions of K+ ions were determined experimentally using anomalous scattering, wherein cesium was substituted for potassium during crystallization. Four K+ ions (K1, K2, K3, and K7) show significant cesium anomalous density while the remaining three were modeled into the structure based on high B factors. For the latter sites, we observed very strong experimentally derived electron density. Diffraction data from crystals containing K+ collected at the CuKa wavelength showed weak K+ anomalous diffraction. However, the current data still cannot rule out other possible explanations for electron density at K4, K5, and K6. The Mg2+ and K+ ions appear only to support the formation of the global RNA structure by forming contacts around its periphery, whereas the four Co2+ ions are centrally located. Notably, the nucleotides involved in forming contacts with K+ are not well conserved, which supports our conclusion that potassium ions play only a supportive role in aptamer formation. Indeed, in-line probing data with a NiCo RNA construct reveal that potassium alone does not trigger the structural changes brought about by Ni2+ or Co2+ (Figure S5B). A key type of divalent cation-RNA interaction in the structure involves the N7 position of several highly conserved guanines (Figure 4; G45, G46, G87, and G88), which are the most nucleophilic of RNA functional groups. The use of these guanine N7 positions might contribute to metal ion selectivity, as these functional groups are likely to bind more strongly to intermediate ions rather than to hard ions, such as Mg2+ (which prefers to coordinate hard anions such as oxygen). For example, in stark contrast to NiCo, a member of a Mg2+-sensing riboswitch class (ykoK RNAs or Mg2+-I riboswitches) employs mainly nonbridging phosphate oxygen atoms to bind Mg2+ ions (Dann et al., 2007; Wakeman et al., 2009). At the base of P2, a fourth Co2+-binding site (C4) is formed in part by G18. Nucleotides from both P1 (G15) and P2 (A41) complete the coordination sphere around cobalt C4 (Figure 4D). Also, in the vicinity of cobalt C4, a Mg2+ ion makes contacts with the phosphodiester backbone of nucleotides 39–41, which likely stabilizes an internal bulge formed by these nucleotides to permit formation of a base triple comprised of nucleotides C20, G39, and G42. Based on its location, it is possible that cobalt 4 aids in positioning P2 with respect to P1. This possibility is supported by the observation that nucleotides G39 to A41 undergo modulation during in-line probing (Figure S3). Cobalt C4 makes fewer contacts with conserved nucleotides and generally shows low occupancy and relatively weak anomalous density (5s) compared to the 12s anomalous density observed for cobalt 1, 2, and 3 (Figures 3D–3F and S4). Hence, we cannot rule out the possibility that this site may be occupied by other metal ions in solution such as potassium or magnesium and hence may not play as critical a role as C1, C2, and C3 for NiCo function. NiCo Displays Cooperative Binding of Metal Ions The clustering of the Co2+ binding sites is likely to be responsible for the cooperative binding characteristic observed in our

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Figure 3. Crystallographic Model of a NiCo in Complex with Co2+ Ions (A) Secondary structure diagram of Eba NiCo according to the crystallographic model. Seven nucleotides were added at the 50 and 30 termini (black) to facilitate crystallization. Two nucleotides in the L4 loop (black) were not modeled due to insufficient electron density. Helices P1-P2 (pink) and P3-P4 (gray) form a ‘‘H’’shaped structure stabilized at the junction by four Co2+ ions. Antiterminator nucleotides (cyan) and long-range interactions (dashed) are indicated. Inner sphere (filled symbols) and water mediated (open symbols) contacts to different metals are shown in key. (B) Crystal structure of NiCo shows two sets of coaxially stacked helices: P1-P2 (pink) and P3-P4 (gray). Interhelical nucleotides coordinate four Co2+ ions (green). Anti-terminator nucleotides 78 to 98 (cyan) are sequestered within P4 and P1, making direct contacts with Co2+ ions. Seven K+ (dark blue) and two Mg2+ (light blue) ions are bound to NiCo. (C) Nucleotides that undergo modulation by in-line probing (red) lie at or near the Co2+ binding sites. (D) A Co2+ anomalous difference Fourier map contoured at 4.5s (yellow mesh) reveals four Co2+ ions (green). C1, C2, and C3 reside on a plane perpendicular to the long axis of NiCo (black disk). (E and F) Side and top view of the molecule shows a highly splayed out structure with minimum long-range contacts. Also see Figure S3 for in-line probing of Eba NiCo RNA and Table 1 for crystallography data collection and refinement statistics.

biochemical assays. The crystal structure exposes a network of molecular interactions between individual Co2+ binding sites (Figure 5A). For example, G87 simultaneously coordinates cobalt 1 through its N7 group and cobalt 2 via its ribose oxygen. Similarly, G45 coordinates cobalt 2 via water-mediated outer-sphere

interactions while also coordinating cobalt 3 via its N7. Thus, binding at one of these sites should stabilize the divalent metal ion bound at the adjoining site. This mechanism is supported by the fact that nucleotides proposed to participate in interconnectivity of the metal sites (G45, G46, G87, and G88) are strictly

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stitutions of the N7 positions of G86, G87, and G88, which we anticipated would have different effects on Co2+ binding based on their different structural contexts. Band intensities at four key sites (G46, G45, C40, and A14) that all undergo robust changes in the wild-type (WT) unimolecular aptamer (Figure S3) were monitored. As expected, the bimolecular aptamer formed by all-natural nucleotides, and the bimolecular aptamer formed with 7-deaza G86, both yield binding results (Figure 6B) that are similar to that of the WT unimolecular construct. However, the construct formed with 7-deaza G87 yields a different in-line probing pattern wherein nucleotide G46 fails to undergo a normal level of Co2+-mediated reduction in spontaneous cleavage (Figure 6C). This result indicates that the 7-deaza G87 alteration, which is predicted to intimately form the cobalt 1 binding site, also affects Co2+-binding at adjacent cobalt site 2. This chemical change, however, does not appear to adversely affect the more distal cobalt 3 binding site, as evidenced by the observation of typical modulation of nucleotides forming this site (G45 and A14) (Figure 6B). Similarly, the presence of 7-deaza G88 in the bimolecular construct causes a substantial loss in binding affinity of the cobalt 2 site and at the adjoining cobalt 3 site as determined by the changes in in-line probing band intensities of nucleotides that comprise these sites. The 7-deaza G88 change to cobalt 2 also disrupts the normal Co2+-dependent modulation of nucleotide C40 (Figure 6C), demonstrating that adverse effects on one metal binding site propagates to distal parts of the aptamer. However, the modest functional group changes used in our constructs are not sufficiently disruptive to completely eliminate Co2+ binding at all sites. Regardless, our findings are consistent with the hypothesis that a network of interactions links the metal binding sites to form NiCo riboswitches with cooperative binding characteristics.

Table 1. Data Collection and Refinement Statistics NiCo + Cobalt + Potassium

NiCo + Cobalt + Cesium

I 422

I 422

Data Collection Space group Cell dimensions a, b, c (A˚)

95.8, 95.8, 229.9

96.24, 96.24, 233.4

a, b, g ( )

90, 90, 90

90, 90, 90

Wavelength (A˚)

0.9772

1.90745

Resolution range (A˚)

50 - 2.64

50- 3.1

Unique reflectionsa

16,067 (779)

19,011(942)

a

99.3 (94.5)

99.7 (100)

Rsym (%)a

0.069 (0)

0.09 (0.43)

I/s(I)a

41.1 (1.9)

10.2 (1.8)

CC*

1.0 (0.93)

0.99 (0.97)

0.03 (0.48)

0.06 (0.27)

Completeness (%)

Phasing Number of sites

4

Initial figure of merit

0.22

After density modification

0.857

Refinement Resolution (A˚)a

33.8 (2.64)

# reflections work/ test set

22,654/2,252

Rfactor / Rfree (%)

19.3/22.8

Rmsds Bond lengths (A˚)

0.005

Bond angles ( )

1.18

Average B factors (A˚2)

35.5

RNA

35.8

Ligands

34.7

Solvent

30.7

Number of atoms

2,109

RNA

1,993

Co

4

K

7

Mg

2

Sr

1

Glycerol

14

water

88

a

Values for the highest resolution shell are in parentheses.

conserved among NiCo homologs (Figures 1A and 5B), suggesting that cooperative ligand binding is maintained through evolution. To provide additional support for the specific Co2+ binding sites observed by X-ray crystallography, and to further assess the source of cooperativity, we conducted in-line probing analyses with a bimolecular construct of the E. bacterium RNA wherein a series of functional group changes (Figure 6A) were evaluated. Specifically, we assessed the impact of carbon sub-

Control of Gene Expression by NiCo Many riboswitches bind their cognate ligands to adopt a structure that controls formation of a downstream intrinsic transcription terminator hairpin (Breaker, 2011; Mandal and Breaker, 2004). Similarly, we predict that the NiCo RNA precludes formation of an overlapping intrinsic terminator stem when ligand is bound (Figure 7A). This is supported by in vitro transcription assays, which confirm that the riboswitch promotes production of full-length transcripts only upon the addition of Co2+ or Ni2+ (Figures 7B and 7C). This effect depends on the aptamer domain retaining its conserved nucleotides and secondary structure features. For example, antitermination is lost for an M3 mutant that disrupts the P2 stem of the RNA (Figures 7B and 7C). A similar effect was observed for other NiCo representatives where Co2+ and Ni2+ induce antitermination at significantly lower concentrations than Mn2+ (Figure S6). Antitermination is lost for mutants M4 and M5 that target conserved interhelical nucleotides (Figure S6). However, mutations in conserved nucleotides are rendered ineffective if the terminator stem is weakened (as in the M5 mutation), resulting in full-length transcripts independent of metal concentration (Figure S6). Similar to metal binding, the in vitro regulatory response also exhibits cooperative behavior, which indicates that the binding characteristics of the independent aptamer are retained in the full-length riboswitch.

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Figure 4. Co2+ Binding Sites in a NiCo Aptamer (A–D) Co2+ ions bind NiCo in octahedral coordination sites. Metal-coordinating nucleotides and water molecules (blue spheres) are indicated. (E) An experimental SAD electron density map contoured at 2.5s (blue) corresponding to the NiCo crystallographic model is shown. A cobalt anomalous fourier map contoured at 4.5s (yellow) helped identify Co2+ ions (green sphere) unambiguously in the structure. Also see Figure S4 for more information on cobalt site electron density.

To address the function of NiCo in the cellular context, we examined a representative located upstream of a putative cation efflux gene (COG0053) of Clostridium scindens. C. scindens cells were cultured anaerobically in rich media containing varying amounts of extracellular Ni2+ and then harvested for quantification of COG0053 transcript abundance. Under these conditions, increasing levels of Ni2+ led to increased COG0053, although control transcripts were not affected (Figure 7D). Similarly, addition of Ni2+ but not Mn2+ or Zn2+ led to increased abundance of a gene downstream of the Clostridium cellulolyticum NiCo (Figure S6E). These aggregate findings are all consistent with our hypothesis that NiCo RNAs are Co2+- or Ni2+-dependent riboswitches. DISCUSSION NiCo RNAs are highly selective sensors of specific divalent transition metals, and these RNAs accomplish this difficult task despite an intracellular milieu containing far greater abundance of other cations such as Mg2+, Zn2+, and Ca2+. Previously, in vitro evolution was used to create allosteric hammerhead ribozymes that are activated by certain heavy metal ions (Zivarts et al., 2005). However, these engineered ribozymes are activated by Mn2+, Fe2+, Co2+, Ni2+, Zn2+, and Cd2+ to a similar

extent, whereas the specificity of NiCo riboswitches is far greater. Therefore, NiCo riboswitches are akin to metalloregulatory proteins, which show incredible specificity in their response to certain metals. For metalloregulatory proteins, this specificity derives in part from precise metal site geometry and coordination chemistry, as well as the differential bioavailability of metal ions (Giedroc and Arunkumar, 2007). For NiCo riboswitches, this geometry is created in part by the positioning of nucleophilic groups from evolutionarily conserved guanine nucleotides. Also, the overall splayed, non-compacted arrangement might help NiCo aptamers sense Ni2+ or Co2+ in a vast amount of competing cytoplasmic cations. If the global structure was susceptible to cation-induced compaction, the riboswitch might be inappropriately triggered by the general shielding of negatively charged phosphates by Mg2+ or K+ ions. For example, the Mg2+-I riboswitch exhibits dramatic compaction upon addition of divalent Mg2+ (Dann et al., 2007; Wakeman et al., 2009). Moreover, the dramatic Mg2+-induced compaction of the Mg2+-I riboswitch is key to its regulation of transcription termination. In contrast, size-exclusion chromatography analysis of NiCo reveals negligible compaction in response to increasing amounts of Mg2+ (Figure S5A). Similarly, the addition of Co2+ does not trigger significant NiCo compaction. These

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Figure 5. An Interconnected Network of Molecular Contacts between Co2+ Sites in a NiCo RNA (A) Co2+ ions (green) bound to NiCo are coordinated by interhelical nucleotides from diametrically opposite sides of the RNA. G87 coordinates cobalt 1 via N7 and cobalt 2 via its ribose oxygen. G45 coordinates cobalt 2 via water mediated contacts with a ribosyl oxygen and cobalt 3 via its N7. These long-range connections (pink dashed lines) extend from A14 to G86 via G45 and G87, connecting three of the Co2+ sites. Coordination distances are listed in Table S1. Also see Figure S5 for global structural changes in a NiCo RNA. (B) Secondary structure schematic summarizing the connectivity of Co2+ ions with interhelical residues. Co2+ coordinating nucleotides are the most tightly conserved in the NiCo sequence.

observations are consistent with a pre-organized NiCo structure, whose overall arrangement may be partially resistant to nonspecific effects caused by the dynamics of abundant intracellular metals.

E. bacterium bimolecular NiCo aptamer

A B A G C G 40 G 60 C GCA P4 A GC G C U GCC A C A GAUg No 46 A G U A 45 Modulation G AC C A G U A A C G G 86 U G U C U A c 5’ G 80 A 20 P2 14 7-deazaG C 14 87 G G A G C A G 88 90 P1UC GA 12 A U Eba 1 A U Eba 2 g c 5’ g c 10 40 C 46 G

C

A G C G P3 U C G GG

KD (10 -6 M)

A

WT 7-deaza G87

Band intensity (normalized)

7-deaza G88

1.0

Metal ions serve many essential roles but are toxic in excess, necessitating mechanisms for controlling their homeostasis. Metalloregulatory proteins accomplish this task by allosterically coupling metal binding sites to control of gene expression. These

Eba 2 Construct WT

7-deaza 7-deaza 7-deaza G86 G87 G88

8 6 4 2

0.5

Nucleotide

0 -7

-6

-5

log c (Cobalt, mM)

G46 G45

Figure 6. Effects of Functional Group Changes on Co2+ Binding by a Bimolecular NiCo Riboswitch Aptamer (A) The bimolecular aptamer derived from E. bacterium NiCo. Complex formation between RNA Eba 1 (black) and chemically altered RNA Eba 2 (gray) was favored by increasing the base-pairing potential of stems P1 and P4 by the addition of several non-native nucleotides (lowercase letters). Nucleotides G86, G87, and G88 were independently modified as denoted, and circled nucleotides were monitored for spontaneous cleavage using in-line probing assays. (B) Plot of the KD values for Co2+ binding by the bimolecular aptamer formed from various Eba 2 constructs. Values were estimated from the modulation of in-line probing bands at four different nucleotides as denoted. (C) Plot of the in-line probing band intensity for various Co2+ concentrations at sites G46 and G40 for the bimolecular aptamer formed using WT Eba 2 RNA or wherein G87 or G88 were replaced with N7-deaza nucleobases.

C40 A14

-4

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M3 GG GG 40

A G C G P3 U C G GG

G CA GC GC C A GGUC AU G AC C A G U A A A C G G A P2 20 C G

5’

B

_

AG U P1C A G U AC AA

C A A E. bacterium G C czcD leader G G 60 C P4 A U UA A AC A GAUG C A U G UC UAC AC GC G 80 G G C A A G A GGGUGUC U AC A 10 U U UC C A C AGA UGU U U U UU U G terminator U A U AUUC C AC AG AUGU U U UUU 3’

% FL

A

Fold Increase in Transcript

T FL

M3

T

[Co 2+ ] FL T

M3

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D

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60

[Ni 2+ ]

WT

_

80

FL

20 COG0053 GAPDH

10

0

0

T

10

20

30

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Nickel (mM)

Figure 7. Control of Gene Expression by a NiCo Riboswitch (A) Sequence and predicted secondary structure of the E. bacterium NiCo RNA. Nucleotides in P4 and downstream of NiCo (gray shading) can form an altered base paired terminator structure as shown. (B) In vitro transcription in increasing metal concentrations reveals a Ni2+- and Co2+-dependent increase in antiterminated full-length (FL) RNA transcript and a corresponding decrease in terminated (T) product. M3 mutation that disrupts Co2+ binding exhibits loss of antitermination. (C) Plot of the yield of full-length transcripts versus Co2+ or Ni2+ concentration for WT and M3 templates. The metal ion concentrations at which elongation efficiency of WT is half-maximal (T50) are indicated with dashed lines. The R2 values for the curve fitting analyses are 0.981 and 0.987 for Co2+ or Ni2+ with WT templates, respectively. (D) Ni2+-induced expression of a gene associated with a NiCo riboswitch in Clostridium scindens. Changes in mRNA level of COG0053 and GAPDH genes in cells exposed to various concentrations of NiCl2 were measured. MDH mRNA levels, which are expected to be unchanged by Ni2+ addition, were used to normalize. Each symbol represents the mean of three independent replicates, and error bars represent SEM. GAPDH, predicted glyceraldehyde-3-phosphate dehydrogenase gene. MDH, predicted malate dehydrogenase gene. Also see Figure S6 for additional in vitro transcription data.

proteins associate with their target ion(s) while discriminating against competing metals, which are often in stoichiometric excess. Several classes of signal-responsive riboswitches control gene expression in response to the divalent ion, Mg2+ (Dann et al., 2007; Wakeman et al., 2009). However, these riboswitches act as general sensors of divalent metals and respond to Mg2+ only by virtue of its high intracellular abundance. We report herein the discovery, validation, and structural details of a true metalloregulatory riboswitch class whose members selectively and tightly bind either Ni2+ or Co2+. Therefore, our data demonstrate that RNA has the ability to form binding sites that recognize certain transition metals with high specificity and affinity. From the analysis of this riboswitch class, general principles can be acquired that could lead to discovery of other classes of metal-responsive RNA regulators and that expand understand-

ing of metal-RNA interactions. Indeed, a Salmonella intrinsic terminator hairpin was recently postulated to be affected by Mn2+ (Shi et al., 2014). Moreover, an exceptionally widespread, and structurally conserved, orphan riboswitch has also been recently shown to specifically function as a Mn2+-responsive riboswitch (G. Storz and L. Waters, personal communication). Therefore, the discovery of these respective metal-responsive riboswitches provides convincing evidence that metalloregulation is not only limited to the current families of metalloregulatory proteins, but instead also broadly derives from phylogenetically widespread families of metalloregulatory RNAs. These data together demonstrate that RNA-based metal sensors are abundant in nature and therefore must be considered alongside the families of protein-based metal sensors that have been discovered.

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EXPERIMENTAL PROCEDURES Bioinformatics Covariance model searches were performed as described earlier (Weinberg et al., 2010) and are detailed in the Supplemental Information. RNA Preparation and In-Line Probing To generate in-vitro-transcribed RNA, DNA templates were made by PCR amplification of synthesized oligonucleotides with the appropriate primers. Transcription was carried out using bacteriophage T7 RNA polymerase in 80 mM N-(2-hydroxyethyl)piperazine-N0 -(2-ethanesulfonic acid) (HEPES) (pH 7.5 at 25 C), 40 mM dithiothreitol (DTT), 24 mM MgCl2, 2 mM spermidine, and 2 mM of each nucleoside 50 -triphosphate (NTP). RNA was purified using denaturing (8 M urea) 10% PAGE. The appropriately sized band was excised and minced into 1 mm2 squares, and the RNA was recovered by passive elution. The RNA was subsequently precipitated with ethanol and pelleted by centrifugation. RNA was radiolabeled at the 50 end and used for in line probing assays as described in the Supplemental Information. Chemicals and Oligonucleotides Oligos were purchased from Integrated DNA technologies or synthesized as described in the Supplemental Information. Chemicals were purchased from Sigma. Clostridium scindens strains used in this study were derived from the ATCC 35704 strain, obtained from American Type Culture Collection (ATCC). Transcription Termination Assays DNA templates harboring the riboswitch sequence from E. bacterium carrying its native promoter or from Clostridium cellulolyticum carrying an RpsD promoter were prepared from synthesized oligonucleotides and appropriate primers by PCR. Transcription termination assays were conducted using a method of single-round transcription detailed in the Supplemental Information. In Vivo Gene Expression Assays Clostridium scindens was cultured in Brain Heart Infusion Broth medium (Fluka, Sigma-Aldrich) under anaerobic conditions at 37 C without shaking until early to mid exponential phase (determined by measuring optical density at 600 nm, around 0.15). 3 ml of cell culture was harvested by centrifugation. NiCl2 (Sigma-Aldrich) solutions were prepared in 1003 concentrations and were added to the culture to final concentrations as noted. The cell culture was incubated at 37 C for 1 hr, at which point 3 ml was harvested by centrifugation. The cell pellet was resuspended in TE buffer (10 mM TrisdHCl, 1 mM EDTA, pH 8.0 at 23 C) with 400 mg ml
1 lysozyme (Sigma-Aldrich) and incubated at room temperature for 10 min. Clostridium cellulolyticum H10 was grown anaerobically in ATCC Medium 1368 modified to contain cellobiose as a carbon source in place of cellulose. Cultures were cultured in rubber-stopper-sealed glass bottles at 34 C for 24 hr. An aliquot of cobalt (II) chloride, manganese (II) chloride, zinc (II) chloride or an equal volume of water was added to each duplicate pair of cultures to a final concentration of 200 mM. After 2 hr of additional incubation, cells were isolated by centrifugation. Total RNA isolation, preparation, and quantification are described in the Supplemental Information. Purification of NiCo RNAs for Crystallographic Analysis NiCo RNA was prepared in vitro in milligram amounts from 10 ml transcription reactions. Following separation on a urea-denaturing polyacrylamide gel, the RNAs were extracted, refolded in 10 mM HEPES (pH 7.5) with 150 mM KCl, and concentrated as described in the Supplemental Information. Representatives from different organisms were screened at 10 mg/ml concentration for crystal formation using the Nucleic Acid Mini Screen (Hampton Research). Crystallization and X-Ray Data Collection NiCo crystals for the Eba representative RNA were obtained by hanging drop vapor diffusion, wherein 4 ml of 10 mg/ml NiCo was incubated for 30 min at room temperature in the presence of 2 mM Co2+ chloride and subsequently mixed with 2 ml of crystallization solution (10% v/v methyl pentanediol, 40 mM sodium cacodylate [pH 7.0], 12 mM spermine tetrahydrochloride, 40 mM lithium chloride, and 80 mM strontium chloride) and equilibrated

against 35% MPD in the reservoir. Crystals grew in 24 hr at 20 C. Treatment of crystals for X-ray data collection is described in the Supplemental Information. Structure Determination Phasing for NiCo crystal structure determination was performed using anomalous diffraction from the bound Co2+ ions. Details are in Supplemental Information. ACCESSION NUMBERS Coordinates for the crystal structure of NiCo RNA bound to cobalt ions have been deposited to the RCSB Protein Data Bank with the PDB ID code of 4RUM. SUPPLEMENTAL INFORMATION Supplemental Information includes six figures, one table, and Supplemental Experimental Procedures and can be found with this article online at http:// dx.doi.org/10.1016/j.molcel.2015.02.009. AUTHOR CONTRIBUTIONS Z.W. and T.V. performed bioinformatics searches. K.F., Z.Z., A.R., and T.V. conducted the in vitro biochemical analyses. Z.Z. conducted in vivo expression assays. A.R. performed the X-ray crystallography analyses. All authors contributed to the design of experiments, interpretation of the data, and preparation of the manuscript. ACKNOWLEDGMENTS We thank members of the Winkler and Breaker laboratories for helpful discussions. We thank Jonathan Goodson (University of Maryland) for assistance with growth of C. cellulolyticum and qPCR analysis. We thank Dr. Dominika Borek (UT Southwestern Medical Center) for suggestions and extensive discussions regarding X-ray structure determination. This work was supported by NIH grants GM081882 to W.C.W. and GM022778 to R.R.B. and by a JSPS fellowship for research abroad to K.F. RNA research in the Breaker laboratory is also supported by the Howard Hughes Medical Institute. Received: September 16, 2014 Revised: November 17, 2014 Accepted: February 3, 2015 Published: March 19, 2015 REFERENCES Agranoff, D.D., and Krishna, S. (1998). Metal ion homeostasis and intracellular parasitism. Mol. Microbiol. 28, 403–412. Anton, A., Grosse, C., Reissmann, J., Pribyl, T., and Nies, D.H. (1999). CzcD is a heavy metal ion transporter involved in regulation of heavy metal resistance in Ralstonia sp. strain CH34. J. Bacteriol. 181, 6876–6881. Baker, J.L., Sudarsan, N., Weinberg, Z., Roth, A., Stockbridge, R.B., and Breaker, R.R. (2012). Widespread genetic switches and toxicity resistance proteins for fluoride. Science 335, 233–235. Bouzat, J.L., and Hoostal, M.J. (2013). Evolutionary analysis and lateral gene transfer of two-component regulatory systems associated with heavy-metal tolerance in bacteria. J. Mol. Evol. 76, 267–279. Breaker, R.R. (2011). Prospects for riboswitch discovery and analysis. Mol. Cell 43, 867–879. Ciesio1ka, J., Micha1owski, D., Wrzesinski, J., Krajewski, J., and Krzyzosiak, W.J. (1998). Patterns of cleavages induced by lead ions in defined RNA secondary structure motifs. J. Mol. Biol. 275, 211–220. Cromie, M.J., Shi, Y., Latifi, T., and Groisman, E.A. (2006). An RNA sensor for intracellular Mg(2+). Cell 125, 71–84.

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