Long-range pseudoknot interactions dictate the regulatory response in the tetrahydrofolate riboswitch 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 vitamin witches. These structures displayed different architectures and folate (B9), is an essential cofactor in one-carbon transfer reactions. shared limited similarities regarding details of ligand recognition In bacteria, expression of folate-related genes is controlled by (12). By contrast, structure determination of cobalamin (13) and feedback modulation in response to specific binding of THF and molybdenum/tungsten cofactors (14) riboswitches 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 domain 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- BIOCHEMISTRY pocket adjacent to the junction, where it forms specific hydrogen cleotides (>75% identity), with much fewer nucleotides exhibiting bonds with two moderately conserved pyrimidines. 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 tetrahydrobiopterin 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 acid, 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 gene 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 thiamine 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 GA C G 70 G 40 U U L4 GC P4 CG J2/3 G AU A U 50 J3/4 G C A CG 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 AG G G GA GC UG 20 UG A G 70 AU G 31 CG 80 CG J2/3 A GC A C U A P2 G J4/2 GC UA U UA GC GUG AU 20 GC U A P2 UA P3 AU Nucleotide GC 80 G C 50 P3 CG GU CG identity AU AG CG P2 AU U G A 10 N 97% A U CU90 C G U UA G A J1/2 N 90% G C A U J1/2 J2/1 G C N 75% GCGC 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 CG C G P1 AU P1A U decreased GU 100 G U 100 5´ constant 1 GC 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.