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Home , Lysine riboswitch, Thiamine pyrophosphate, TPP riboswitch

Long-range pseudoknot interactions dictate the regulatory response in the tetrahydrofolate

Lili Huang1, Satoko Ishibe-Murakami, Dinshaw J. Patel1, and Alexander Serganov1,2

Structural Biology Program, Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, New York, NY, 10065

Contributed by Dinshaw J. Patel, July 18, 2011 (sent for review July 9, 2011)

Tetrahydrofolate (THF), a biologically active form of the witches. These structures displayed different architectures and folate (B9), is an essential in one-carbon transfer reactions. shared limited similarities regarding details of ligand recognition In , expression of folate-related is controlled by (12). By contrast, structure determination of cobalamin (13) and feedback modulation in response to specific binding of THF and /tungsten cofactors (14) has been ham- related compounds to a riboswitch. Here, we present the X-ray pered by the low stability of the riboswitch ligands and complexity structures of the THF-sensing from the Eubacterium sir- of the RNA folds. aeum riboswitch in the ligand-bound and unbound states. The The secondary structure of the metabolite-binding domain of structure reveals an “inverted” three-way junctional architecture, the THF riboswitch is predicted to adopt a three-way junctional most unusual for riboswitches, with the junction located far from architecture comprised of four helical segments joined by an the regulatory helix P1 and not directly participating in helix P1 internal loop and a three-way junction (2) (Fig. 1A). Further, formation. Instead, the three-way junction, stabilized by binding the apical loop L3 has the potential to form a long-range tertiary to the ligand, aligns the riboswitch stems for long-range tertiary pseudoknot with the 3′ segment of the internal loop. The most pseudoknot interactions that contribute to the organization of conserved regions of the riboswitch, which are located within helix P1 and therefore stipulate the regulatory response of the and around the internal loop, the three-way junction, and loop riboswitch. The pterin moiety of the ligand docks in a semiopen L3, are predominantly composed of moderately conserved nu- pocket adjacent to the junction, where it forms specific hydrogen cleotides (>75% identity), with much fewer nucleotides exhibiting bonds with two moderately conserved . The amino- significant (>90% identity) and high (>97% identity) conserva- benzoate moiety stacks on a guanine base, whereas the glutamate tion. On the other hand, integrity of helices P2 and P3 is critical moiety does not appear to make strong interactions with the RNA. for high-affinity binding of THF (2). In contrast to other riboswitches, these findings demonstrate that The THF-binding site has not been unambiguously identified the THF riboswitch uses a limited number of available determi- by conventional biochemistry. In-line probing revealed modula- nants for ligand recognition. Given that modern antibiotics target tion of RNA structure upon THF binding in all three conserved folate metabolism, the THF riboswitch structure provides insights regions (2), which, given the ligand dimensions and anticipated on mechanistic aspects of riboswitch function and may help in compaction of RNA after ligand binding, could all contribute manipulating THF levels in pathogenic bacteria. to THF recognition. Deletion analysis suggested that the internal ∣ ∣ ∣ loop and the tertiary pseudoknot most likely support folding RNA structure vitamin B9 coenzyme of the ligand-binding pocket, but are not essential for ligand binding (2). Because the majority of other riboswitches contain he tetrahydrofolate (THF) compounds, biologically active a ligand-binding pocket in a junctional region, the THF-binding Treduced derivatives of folate (vitamin B9), are essential cofac- pocket could reside within a three-way junction as well. However, tors in one-carbon transfer reactions involved in the biosynthesis unlike other riboswitches, the junction is positioned far from the of many critical molecules. Therefore, maintaining an adequate regulatory helix P1 in the THF riboswitch. The removal of the cellular level of THF is of primary importance to all living beings. aminobenzoate and glutamyl moieties in tetrahydrobiopterin In bacteria, THF can be generated from folate transported from (THBP) decreased binding affinity approximately fourfold (2) the environment using a special transport system (1) or synthe- (Fig. 1B), suggesting that the THF-binding site is organized to sized de novo. Expression of folate transport and synthetic genes specifically recognize the pterin moiety and is likely to be com- is controlled by riboswitches that respond to THF and related pact. The riboswitch appears to recognize only the reduced form compounds (2). B Structured mRNA segments termed riboswitches act as both of the pterin moiety, as in THF and 5-formyl-THF (Fig. 1 ), direct sensors of cellular metabolites and effectors of the regula- whereas oxidation of the pterin moiety, as in folic , eliminated tory response in all three kingdoms of life (3, 4). Typically, specific binding. binding of a cognate metabolite stabilizes the metabolite-bound conformation of the sensing domain of the riboswitch and, Author contributions: L.H., D.J.P., and A.S. designed research; L.H., S.I.-M., and A.S. through formation of the regulatory helix P1, directs the folding performed research; L.H., D.J.P., and A.S. analyzed data; and L.H., D.J.P., and A.S. wrote of the adjacent expression platform that carries signals for tran- the paper. scriptional or translational machineries. If the metabolite concen- The authors declare no conflict of interest. tration does not reach a threshold, the riboswitch adopts an Data deposition: The atomic coordinates and structure factors have been deposited in alternative conformation, resulting in an opposite effect on the Protein Data Bank, www.pdb.org [PDB ID codes 3SUY (ligand-free THF riboswitch), 3SUX (riboswitch bound to THF), 3SUH (riboswitch bound to 5-formyl-THF)]. expression. Riboswitches respond to various types of metabolites; 1To whom correspondence may be addressed. E-mail: [email protected], however, the most widespread and abundant riboswitches, pre- [email protected], or [email protected]. sently counting nine classes, are selective to protein coenzymes. 2Present address: Department of Biochemistry, New York University School of Medicine, To date, X-ray structures have been solved for pyropho- New York, NY 10016. – S sphate (TPP) (5 7), FMN (8), and three classes of -adenosyl- This article contains supporting information online at www.pnas.org/lookup/suppl/ (L)-methionine (SAM-I, SAM-II, and SAM-III) (9–11) ribos- doi:10.1073/pnas.1111701108/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1111701108 PNAS ∣ September 6, 2011 ∣ vol. 108 ∣ no. 36 ∣ 14801–14806 Downloaded by guest on October 1, 2021 ACL3 DE AA GA L4 G G AG C G 70 G 40 U U L4 GC P4 GC J2/3 G UA A U 50 J3/4 G C A GC J3/4 P4 G C G U J2/3 A U G 5F-THF A C A G C J4/2 G A P3 U G J3/4 G 60 A A G C J4/2 C A P4 L4 5F-THF G G 28 C C GA G 30 G A C C A 58 C U U 60 C A A GA G G GA CG GU 20 UG A G 70 UA G 31 GC 80 GC J2/3 A CG A C U A P2 G J4/2 CG AU U AU GC UG G UA 20 CG U A P2 AU P3 UA Nucleotide CG 80 G C 50 P3 GC UG GC identity UA GA GC P2 UA U G A 10 N 97% A U UC 90 C G U AU G A J1/2 N 90% G C A U J1/2 J2/1 G C N 75% CG GC J2/1 L3 A 10 A L3 G U U 90 U G C J1/2 G C G G A J2/1 Scission on U A C 40 THF binding G CG G C A U A U increased GC C G P1 UA P1A U decreased UG 100 G U 100 5´ constant 1 CG 1 G C P1 5´ 3´ 5´ 3´ 5´ 3´ 3´

B O FG THF O H 5-Formyl-THF THBP O- HO O N O O OH H H H H H H N 10 N R N HN 5 N HN 5 HN 8 H O O- 8 OH H N NN H2N NN H2N NN 2 H H H 5´ 5´ Tetrahydropterin 5´ p-Amino Glutamyl P1 P1 benzoate 3´ 3´ 3´

Fig. 1. Sequence and structure of the E. siraeum THF riboswitch. (A) Secondary structure schematics of the E. siraeum THF riboswitch used for crystallization and projected results of in-line probing on the THF riboswitch from Alkaliphilus metalliredigens (2). Nucleotide conservation in the THF riboswitch family and THF-induced modulations in in-line probing are explained in inset. Dashes and solid lines indicate Watson–Crick base pairs observed in the crystal structure. Solid circles and dashed lines depict noncanonical hydrogen bonds. Long-range pseudoknot interactions are shown according to the structure. Shading cor- responds to the colors used for secondary structure elements in other figures. To facilitate crystallization, noncanonical base pairs were converted to their Watson–Crick counterparts by U14A, U65C, and U85A mutations. (B) Chemical structures of THF, 5-formyl-THF, and THBP. Gray shading highlights protonation sites in the pterin moiety. (C) Crystal structure-based schematic of the RNA fold of the THF riboswitch. The bound 5-formyl-THF (5F-THF) is in red. Key tertiary stacking interactions are shown as blue dashed lines. Red squares indicate >97% conserved nucleotides. (D) Composite crystal structure of the THF riboswitch in the bound state in a ribbon representation. (E) Same structure, back view. (F) Splayed apart strands of helix P1 in a single riboswitch molecule. (G) Formation of helices P1 with segments from two symmetry-related riboswitch molecules.

To define the architecture of the THF riboswitch, understand seven nucleotides on the 5′ side of helix P1 are paired with the its ligand recognition principles, and deduce insights into the 3′ segment of helix P1 from the adjacent RNA (Fig. 1 F and G and mechanism of the THF riboswitch action, we determined crystal Fig. S1). Because metabolite binding is expected to facilitate the structures of the THF riboswitch sensing domain in the free and organization of the regulatory helix P1, the individual riboswitch ligand-bound states. These structures identified the unique archi- molecules in the crystal lattice could be considered to be in the tecture of the THF riboswitch, including its three-way junction, state preceding formation of P1. On the other hand, a composite pseudoknot, and unusual location of the ligand-binding pocket, molecule that contains helix P1 made from segments that belong thereby defining long-range tertiary interactions that account to two riboswitch molecules most likely reflects the near-native for the stabilization of the regulatory helix and the modulation conformation of the ligand-bound sensing domain. A similar, of . though smaller domain swap, has recently been documented in the structure of the S-adenosyl-(L)-homocysteine riboswitch (15). Results Although the domain swap in the THF riboswitch structure does Global Fold of the Sensing Domain of the THF Riboswitch. Our efforts not represent a natural situation, the structure suggests that helix have focused on the THF riboswitch embedded in the 5′-untrans- P1 could be relatively easily melted, thereby emphasizing the fine lated region of the Eubacterium siraeum DSM gene that encodes balance between regulation-relevant alternative conformations of a protein with a homology to the FolT protein, a component this region. of the folate transporter (2). To this end, we have determined the structure of the sensing domain of this riboswitch in the Three-Way Junction Defines the Architecture of the Riboswitch. The ligand-bound state at 2.65-Å resolution (Materials and Methods three-way junction of the THF riboswitch forms a compact struc- and Table S1). The global architecture of the THF-bound ture stabilized by multiple interactions (Figs. 1A and 2A) that RNA conforms to a three-way junctional fold predicted by com- define the geometry of the junction and contribute to the parallel parative sequence analysis (2). The fold comprises helical stem orientation of stems P2 and P3∕L3. The most critical element of P2, connected by joining regions J2∕3,J4∕2, and J3∕4 with hair- the junction is a reverse turn adopted by the J2∕3 region (Fig. 2A). pins P3∕L3 and P4∕L4 (Fig. 1 C–E). Stem P2 coaxially stacks on On the one hand, this joining region extends helix P2 by forming P4 and is aligned parallel to P3, which hosts the ligand-binding the U23•G74 (Fig. 2B and Fig. S2A) and G22-C75 (Fig. 2B and pocket. As anticipated (2), the terminal loop L3 forms tertiary S2B) base pairs, which contribute to the upward orientation of pseudoknot interactions with the joining region J2∕1. hairpin P4∕L4 through continuous stacking of the G73, G74, In the crystal lattice, each riboswitch molecule is involved in C75, G76, and U78 from the P4, J4∕2, and P2 regions (Fig. 2B). a domain swap with a neighboring molecule such that the last On the other hand, three consecutive purines of J2∕3, A25, A26,

14802 ∣ www.pnas.org/cgi/doi/10.1073/pnas.1111701108 Huang et al. Downloaded by guest on October 1, 2021 A BCG74 A24 A61 J2/3 C62 P4 G73 A61 A25 J3/4 A60 A60 A25 G59 A61 U23 G73 J4/2 C58 G22 A26 U28 G59 G27 A57 G74 C75 U19 A26 G27 G76 P3 P2 C21 U28 5-Formyl-THF G77 A79 C58 G29

D G22 E F G27 A25 G59 A26 G20 C21

A60 G77 U78 C75 G76 G76

Fig. 2. Molecular details of the three-way junction. (A) Overall view of the three-way junction with bound 5-formyl-THF. The in-line cleavage propensities of underlined nucleotides are modulated upon THF binding in the A. metalliredigens riboswitch (2). (B and C) Alternate side views of the junction. Putative hydrogen bonds contributing to tertiary interactions and ligand recognition are shown in black and red dashed lines, respectively. (D–F) Top views of the junctional tertiary interactions. Each panel corresponds to a section of the ligand-bound junction as viewed from top to bottom.

and G27, contribute to the downward orientation of hairpin riboswitch recognition in THF (2). THF and its many analogs P3∕L3 through stacking and hydrogen bonding with J3∕4 and are easily cleaved at the C9-N10 bond (20), therefore cocrystals

a top pair of P3 (Fig. 2 B and C). This part of the structure of the riboswitch with THF, 5-methyl-THF, and 10-formyl-THF BIOCHEMISTRY involves two stacks, G59-A60-A25 and A26-G27-U28, stabilized contained mostly the tetrahydropterin moiety bound to the RNA by extensive interactions of A25, A26, and G27 with a sugar and a (Fig. 3B). In this assigned position, the N2-N1-N8 and N2-N3 C base of A60 and a base of G59 (Fig. 2 ). In addition, A25, A26, edges of pterin make hydrogen bonds with the Watson–Crick and G27 interlink the junction through interactions with the edges of moderately conserved U28 and C58 (Fig. 3 A and B). minor groove of the G22-C75 (Fig. 2D and Fig. S2B), C21-G76 (Fig. 2E and Fig. S2C), and G20-U78 (Fig. 2F and Fig. S2D) base The N5 edge of the ligand remains vacant, supporting the obser- pairs, thereby forming A25•(G22-C75) base triple (Fig. 2D), vation that N5-modified THF derivatives retain binding affinity A60•A26•(C21-G76) base quadruple (Fig. 2E), and tetranucleo- to the riboswitch (2). The donation of a hydrogen bond from tide arrangement (Fig. 2F) across the junction. N8 of the ligand to O2 of U28 depends on the protonation status The G59-A60-A61 region of J3∕4 appears to be the easiest for detachment from the junctional core. This segment does not A U28 stack on helices P3 and P4 and is held in place mostly by stacking with A25 from J2∕3 (Fig. 2C) and noncanonical hydrogen bond- ing which includes a type I A-minor triple A61•(U23•G74) 1 3 8 G77 (Fig. S2A), as well as interactions with the base and sugar edges C58 5 of A26 (Fig. 2E) and G27 (Fig. 2F). However, the packing of 3∕4 J to the junctional core is essential for stabilization of the 5-Formyl-THF junction and parallel alignment of P2 and P3. Otherwise, P3 could have more freedom because its directionality would be primarily dependent on a single stacking interaction between U28 and G27 (Fig. 2C). The critical role of region J3∕4 is emphasized by evolu- B U28 tionary conservation of G59, A60, and their pairing partners in J2∕3 (Fig. 1A).

1 3 8 G77 Ligand Binding Stabilizes the Junction. Stabilization of a junctional C58 5 region by ligand binding is a recurrent theme in riboswitch (16, 17). The THF riboswitch joins this common trend except that Pterin of THF its ligand binds adjacent to the junction, a feature also observed C for the glycine riboswitch (18, 19). Projection of the THF-induced G59 modulations (2) (Fig. 1A) on the riboswitch structure (Fig. 2 A and C) shows that THF binding reduces flexibility of J3∕4 and C58 adjacent U28 and G77, and likely facilitates pairing of this seg- U28 ment, predicted to be crucial for junction stabilization. To exert these functions, the ligand binds in the vicinity of J3∕4, as sug- G29 G77 2F − F F − F A57 gested by the elongated o c and o c maps of the bound 5-formyl-THF, that are positioned above the noncanonical • Fig. 3. Ligand recognition by the THF riboswitch. The ligand-binding pock- G29 A57 base pair, flanked by U28 and C58, covered by G59 ets of the 5-formyl-THF-bound (A) and THF-bound (B) structures superposed and extended toward G77 (Figs. 2 A and C and 3A). The observed F − F with the omit o c electron density map (pink) calculated prior to the density between U28 and C58 was assigned to the 5-formyltetra- addition of ligands and contoured at 3σ level. (C) Ligand-binding pocket 2F − F 1σ hydropterin moiety, which is the only primary determinant of in the unbound state. Brown map shows refined o c map at level.

Huang et al. PNAS ∣ September 6, 2011 ∣ vol. 108 ∣ no. 36 ∣ 14803 Downloaded by guest on October 1, 2021 of N8 and requires a reduced form of the ligand (Fig. 1B), in in the map of this structure, which was found to be very similar to agreement with binding experiments (2). Notably, the pterin moi- the ligand-bound structure (Fig. 3C), as previously described for ety buries only about 75% of its surface, reminiscent of in vitro lysine (23, 24) and glycine riboswitches (19). As discussed earlier selected RNA aptamers (21). (19), similarity between ligand-free and bound structures suggests The docking of the tetrahydropterin into the minor groove of that, in the absence of the alternative pairing sequence from the P3 holds C58 and U28 together and provides a partial stacking downstream expression platform region, the ligand-free sensing with G29 and G59 (Fig. 2A), thereby contributing to the packing domain can adopt a near ligand-bound conformation and retain it of J3∕4 into the junctional core. Because the affinity of THBP in the crystal. Although the moderate resolution of the ligand- for the riboswitch was reduced by only a small extent (2), the free structure does not allow drawing of definitive conclusions, interactions between RNA and tetrahydropterin moiety appears observation of several small shifts in the ligand-binding pocket to be sufficient for stabilization of the top part of P3 and the (Fig. S2F) may be indicative of local flexibility of nucleotides entire junctional region. The benzoate ring of 5-formyl-THF was prior to ligand binding, in agreement with the solution data (2). found stacked on the G77 base (Fig. 3A), thereby explaining the observed modulation of this nucleotide upon THF binding and Long-Range Pseudoknot Interactions Stabilize Regulatory Helix P1. reduced binding affinity in THBP (2). In the several structures Parallel-aligned stems P3∕L3 and P2 place the apical loop L3 of proteins bound to THF analogs, the benzoate ring also stacks and the asymmetric internal loop J1∕2 − J2∕1 in proximity to on aromatic amino (22) (Fig. S2E). In the THF riboswitch, each other (Fig. 1 C–E). The difference in length of the 5′ and the density for the glutamyl moiety is not as well defined as for 3′ segments of the asymmetric loop causes disruption of the he- the other parts of the ligand. This observation suggests more than lical conformation, bulging out of several nucleotides from J2∕1 one conformation for this moiety and highlights the lesser con- and bending of this region toward L3 (Fig. 1D). As a result of tribution of the glutamyl moiety for RNA binding. Nonetheless, tight juxtaposition, loops L3 and J1∕2 − J2∕1 can form long-range the main-chain carboxyl of glutamyl may be positioned within a tertiary interactions (stereo views in Fig. 4 A and B). These inter- hydrogen bond distance from N2 of G77 as in Fig. 3A, which actions include four consecutive base pairs G41-C94, G42•U93, shows the glutamyl conformation that accounts for the observed G43-C92, and A44-U91, two stacking interactions G41/A7 and electron density maps. Thus, the THF riboswitch appears to G40/(G8-C89), and several base–ribose, ribose–ribose, and ri- evolve the THF-binding determinants that eliminate cross-bind- bose–phosphate hydrogen bonds involving A7, G8, A38, C39, ing with glutamate, which is abundant in cells. Likewise, proteins G40, G41, G88, and C89 (Fig. 4 A and B). The consecutive rarely provide an extended interface for the recognition of the tertiary base pairs adopt a helical conformation, which, through THF glutamyl moiety. stacking with the A7•G95 base pair, propagates to P1 forcing To further validate the location of the ligand and identify the collinear alignments of the P3∕L3 hairpin and helix P1 ligand-induced changes, we determined the ligand-free ribos- (Fig. 4 A–C). Therefore, the tertiary base pairing appears to witch structure at 3.2-Å resolution. The ligand density is missing organize the bottom part of the asymmetrical loop and facilitate

A G48 P3 G48 P3 C P2 P2 C87 U47 U47 P2 G88 G88 U46 A38 U46 A38 G11

C89 C89 L3 G45 C39 L3 G45 C39 A10 A10 U91 U91 G8 G8 A44 G40 J1/2 A44 G40 J1/2 C92 A90 C92 A90 P1 P3 U93 A7 G6 U93 A7 G6 U91 G41 G41 U46

C94 P1 C94 P1 L3 J2/1 J2/1 C96 C96

C50 C50 B P3 P3 D C39 G12 G12 P2 G48 P2 G48 G37 G37 G88 L3 G88 L3 G11 G11 G8 C89 C89 A10 A10 G8 G45 G8 G45 U91 U91 G40 G40 G40 A44 A44

J1/2 G43 J1/2 G43 G41 A7 A7 A7 G41 G42 U93 G41 G42 U93

C94 C94 G6

J2/1 J2/1

Fig. 4. Formation of tertiary pseudoknot interactions. Front (A) and back (B) stereo views of the tertiary interactions between L3 and J1∕2–J2∕1 loops. Under- lined nucleotides are modulated upon THF binding in in-line probing of the A. metalliredigens riboswitch (2). The A38•G48 base pair is not formed in the 5-formyl-THF-bound structure. (C) Top view of the tertiary interactions region showing collinear alignment of the P3∕L3 stem and helix P1. The P2 and P3∕L3∕P1 stems of the riboswitch are highlighted by dashed ovals. (D) Stacking interactions involved in the formation of tertiary interloop contacts and the helix P1 stabilization.

14804 ∣ www.pnas.org/cgi/doi/10.1073/pnas.1111701108 Huang et al. Downloaded by guest on October 1, 2021 the formation of the regulatory helix P1. Not surprisingly, purines In contrast to most riboswitches and similar to the Mg2þ sen- from loop L3 involved in this base pairing are highly conserved sor, the THF riboswitch contains a three-way junction on the among THF riboswitches, whereas the complementary region periphery, away from helix P1, and like the Mg2þ sensor and J2∕1 has evolved to contain pyrimidines (Fig. 1A). The other ter- SAM-I riboswitch, exploits the concept of long-range tertiary tiary interactions, involving stacking of G40 on G8-C89 base pair interactions for helix P1 stabilization (Fig. 5C). However, the (Fig. 4D) and hydrogen bonds with the backbone (Fig. 4 A and B), THF riboswitch structure establishes that the ligand does not anchor the loop regions to each other and reinforce the tertiary make direct contact near P1 and binds to a remote pocket located contacts through packing of A38 and C39 with the minor groove next to the junction. In this location, the ligand organizes the of the upper part of J1∕2–J2∕1 (Fig. 4A). The tertiary contacts are flexible junctional “joint” to align helices for the formation of additionally locked up by hydrogen bonding between nucleotides extensive tertiary interactions that stabilize helix P1. Hence, we of P2 (G11 and G12) and P3 (G37 and C50) that close the loops propose that, in the E. siraeum THF riboswitch, helix P1 stabili- (Fig. 4B). These regions, as well as the loop regions adjacent to zation causes the formation of a downstream hairpin that may helices P2 and P3, are built by evolutionarily conserved residues serve as a transcription terminator and possibly facilitate seques- (Fig. 1A) and likely guide the formation of loop conformations tration of the ribosome binding site for the read-through tran- capable of making tertiary contacts, because the majority of loop scripts. In the absence of THF, the riboswitch helices are not nucleotides are flexible in the absence of the ligand (2). properly aligned, thereby either precluding the formation or lim- The tertiary pseudoknot was predicted to be comprised of six iting the strength of the tertiary interactions (Fig. 5C). Despite base pairs (2); however, the THF riboswitch structure revealed recurrent occurrence of packed reversed helices in different only four base pairs, which included the nonpredicted G41- RNAs, including a metabolite-binding ribozyme (28), an inverted C94 base pair and did not include three predicted base pairs. This architecture of the THF riboswitch, where the ligand-bound discrepancy is unlikely to be explained by fraying of the P1 helix junction and the tertiary contact region switched places relative in the structure because the most affected RNA conformation to the regulatory helix P1, is unique for riboswitches. Thus, the should be around the U9-A10 step, where the strands of a ribos- ligand-bound THF riboswitch structure expands on the structural witch molecule splay apart (Fig. 1F) and where the segment principles utilized for stabilization of the regulatory helix P1 and from the symmetry-related riboswitch molecule makes its entry on the molecular mechanisms underlying riboswitch function. (Fig. 1G). Because the J2∕1 segment is one nucleotide longer Biosynthesis of folates has been targeted by antifolate antibio- in the E. siraeum THF riboswitch than in the consensus sequence, tics for treatment of a broad range of bacterial infections over

it is conceivable that long-distance tertiary base pairs may vary the past three decades. Antibiotic resistance has limited the BIOCHEMISTRY among different species. application of these drugs; nevertheless, some older generation antifolates are still in clinical use, while newer inhibitors of the Implication of THF Binding for the Regulatory Mechanism. The vast folate biosynthesis are currently being developed (29). THF majority of riboswitches modulate gene expression through li- riboswitches were found in clinically relevant bacteria (2), where gand-dependant organization of a helical junction which either they predominantly control folate transport. Although THF directly sequesters a regulatory sequence or contributes to pairing riboswitches are unlikely to serve as sole drug targets, our struc- of a switching segment within adjacent helix P1 (17). Most ligands tural data provide critical information on the molecular mechan- stabilize junctions by interactions with the junctional cores, as ism of THF regulation in bacteria, thereby improving our exemplified by purine riboswitches (25, 26), or close to the cores, understanding of folate biosynthesis and its potential manipula- as in the TPP riboswitch (5–7) (Fig. 5A). In addition, junctional tion in pathogens. riboswitch folds are typically reinforced by the long-range tertiary interactions between peripheral elements (Fig. 5A). An alternate Conclusion architectural principle is utilized in the SAM-I riboswitch where a The THF riboswitch structure has uncovered a riboswitch fold short hairpin folds back toward P1, sandwiches a SAM molecule featuring an inverted junctional architecture where the three- between itself and P1, and anchors to P1 via several backbone way junction and the region of the long-range tertiary interactions interactions (11). Such architecture is more pronounced in the have switched places in comparison to the vast majority of ribos- Mg2þ sensor, functionally related to riboswitches (27). In this witches. In this fold, the three-way junction is located far from metallosensor, a three-way junction is located much further from the regulatory helix P1, but is still capable of directing the reg- P1 and therefore P1 is exclusively stabilized by tertiary interac- ulatory response of the riboswitch through the ligand-depen- tions between the region adjacent to P1 and the Mg2þ-bound dent stabilization of long-range tertiary pseudoknot interactions. helical bundle that folds back toward P1 (Fig. 5B). Unlike other riboswitches, the THF riboswitch specifically recog-

Junction A BC Transcription Sensor stabilization termination or RBS sequestering Expression + platform THF THF TPP Helix RBS Start formation Pur (U)6 P1 P1 Mg2+ ? RBS Tertiary contact P1 5' 5' 5' formation Start 5' (U)6

Fig. 5. Regulatory mechanism of the THF riboswitch. (A) General schematic representation of regulatory helix P1 stabilization in junctional riboswitches. Red ovals indicate positions of the bound purines and thiamine in the cognate riboswitches. The switching sequence involved in the alternative base pairing is shown in magenta. The long-range tertiary interactions between peripheral elements are depicted by blue dashes. (B) Model of the regulatory helix stabilization in the Mg2þ sensor (27). Red sphere depicts four critical Mg2þ cations in the tertiary contact area. Other Mg2þ cations implicated in the riboswitch response and located outside of the tertiary contact area are not shown. (C) Proposed regulatory mechanism of the E. siraeum THF riboswitch. Formation of stable helix P1 is achieved through long-range interactions induced by the THF binding (red oval) to the remotely positioned ligand-binding pocket. RBS, ribosome binding site. Start, translation initiation codon. An arrow indicates translation of the downstream gene.

Huang et al. PNAS ∣ September 6, 2011 ∣ vol. 108 ∣ no. 36 ∣ 14805 Downloaded by guest on October 1, 2021 nizes only the small pterin moiety of the ligand. The remaining atmosphere in a glove box filled with . Crystallization of the ribos- portion of the ligand does not contribute significantly to the bind- witch in the unbound state was carried out in a similar manner in a buffer ing affinity, with almost one-third of the ligand (glutamyl moiety) containing 0.1 M Tris·HCl, pH 7.0. For data collection, crystals were flash- not interacting with the riboswitch. Therefore, the THF ribos- frozen in liquid nitrogen. For soaking, crystals were transferred into 2.3 M ½ ð Þ witch may be specific to a family of the THF-related cofactors, as Na-acetate, pH 7.0, supplemented with 200 mM Ir NH3 6 Cl3, and soaked was suggested from the biochemical experiments (2). In addition, for 6 h. Data were collected at 100 K at the 24-ID-C beamline of the Advanced Photon Source and processed with HKL2000 (HKL Research). the pterin moiety binds to a simply organized semiopen binding pocket, which, unlike what has been reported in many ribos- Structure Determination, Refinement, and Analysis. The structure of the THF witches, is adjacent to the junction and is not positioned within riboswitch was determined by multiwavelength anomalous dispersion meth- the junctional core. The combination of these characteristics od using 3.1 Å iridium hexamine data and refined with Phenix (31) (Table S1). makes the THF riboswitch stand out among the known RNA The RNA model was built in Coot (32) and refined in Phenix and Refmac (33) sensors. using a 2.9-Å native dataset. All other structures were refined using the native structure as a starting model (Table S1). The ligand moiety was added Materials and Methods to the model at a later stage based on the experimental and refined maps of RNA Preparation, Crystallization, and Data Collection. RNAs were transcribed in the ligand-bound and ligand-free structures. The contact surface area and vitro and purified by denaturing polyacrylamide gel electrophoresis followed the solvent accessibility were calculated by the PISA Web server (http:// by anion-exchange chromatography (30). Ligands were dissolved in 1 M www.ebi.ac.uk/msd-srv/prot_int/cgi-bin/piserver). Figures were prepared 2-mercaptoethanol under nitrogen. Complexes of the riboswitch with THF with PyMOL (http://www.pymol.org). or analogs (MP Biochemicals and Merck & Cie) were prepared by mixing ca. 0.4 mM RNA with ca. 0.8 mM ligand, diluted threefold using 1 M dithio- ACKNOWLEDGMENTS. We thank the personnel of beamline ID-24-C at the threitol, in a buffer containing 50 mM potassium acetate, pH 6.8, and 5 mM Advanced Photon Source and Dr. Jiamu Du for assistance with data collection. MgCl2. Crystals were grown at 20 °C for 1 wk in the dark using hanging-drop We thank Dr. O. Ouerfelli, Memorial Sloan–Kettering Cancer Center (MSKCC) 2∶1 vapor diffusion method after mixing the complex at a ratio with the for the synthesis of iridium hexamine and Dr. Samuel Danishefsky (MSKCC) reservoir solution containing 0.1 M Na-Hepes, pH 7.5, or Na-Cacodylate, for providing access to a glove box. We thank Merck & Cie for providing pH 6.6, and 3.2–4.2 M Na-formate. In attempts to prevent degradation 5-formyltetrahydrofolate. D.J.P. was supported by National Institutes of of THF, crystals were typically grown in the dark and in an oxygen-free Health Grant GM073618.

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