Metabolite-sensing mRNAs, or riboswitches, specifically interact with small ligands and direct expression of the genes involved in their metabolism. Riboswitches contain sensing “aptamer” modules, capable of ligand-induced structural changes, and downstream regions, harboring expression-controlling elements. We have focused our RNA regulatory research to metabolite-sensing mRNAs, given the new and unexpected role for non-coding RNA as a riboswitch, and because structural-energetics information will be critical for defining allosteric mRNA transitions associated with the modulation of gene expression levels and metabolic homeostasis.
Our laboratory has published the following reviews on riboswitches and ribozymes:
Serganov, A. & Patel, D. J. (2012). Metabolite recognition principles and molecular mechanisms underlying riboswitch function. Ann. Rev. Biophys. 41, 343-370.
Serganov, A. & Patel, D. J. (2012). Molecular recognition and function of riboswitches. Curr. Opin. Struct. Biol. 22, 279-286.
Serganov, A. & Patel, D. J. (2009). Amino acid recognition and gene regulation by riboswitches. Biochem. Biophys. Acta. 1789, 592-611.
Serganov, A. & Patel, D. J. (2007). Ribozymes, riboswitches and beyond: regulation of gene expression without proteins. Nat. Rev. Genetics 8, 776-790. [PubMed Abstract]
Structural and Dynamic Basis for Low Affinity-High Selectivity Binding of L-glutamine by the Glutamine Riboswitch
Naturally occurring L-glutamine riboswitches occur in cyanobacteria and marine metagenomes where they reside upstream of genes involved in nitrogen metabolism. By combining X-ray, NMR and MD, we characterized a L-glutamine-dependent conformational transition in the Synechococcus elongatus glutamine riboswitch from tuning-fork to L-shaped alignment of stem segments. This transition generates an open ligand-binding pocket with L-glutamine selectivity enforced by Mg2+-mediated intermolecular interactions. The transition also stabilizes the P1 helix through a long-range ‘linchpin’ Watson-Crick G-C pair capping interaction, while melting a short helix below P1 potentially capable of modulating downstream readout. NMR data establish that the ligand-free glutamine riboswitch in Mg2+ solution exists in a slow equilibrium between flexible tuning-fork and a minor conformation, similar but not identical to the L-shaped bound conformation. We propose that an open ligand-binding pocket combined with a high conformational penalty for forming the ligand-bound state provide mechanisms for reducing binding affinity while retaining high selectivity.
Ren, A., Xue, Y., Peselis, A., Serganov, A., Al-Hashimi, H. and Patel, D. J. (2015). Structural and dynamic basis for low-affinity, high-selectivity binding of L-glutamine by the glutamine riboswitch. Cell Reports 13, 1800-1813.
Cyclic dinucleotides are second messengers that target the adaptor STING and stimulate the innate immune response in mammals. Besides protein receptors, there are bacterial riboswitches that selectively recognize cyclic dinucleotides. We recently discovered a natural riboswitch that targets 3’,3’-cGAMP, which is distinguished from the endogenous mammalian signal 2’,3’-cGAMP by its backbone connectivity. Here we report on structures of the aptamer domain of the 3’,3’-cGAMP riboswitch from Geobacter in the 3’,3’-cGAMP and c-di-GMP bound states. The riboswitch adopts a tuning fork-like architecture with a junctional ligand-binding pocket and different orientations of the arms are correlated with the identity of the bound cyclic dinucleotide. Subsequent biochemical experiments revealed that specificity of ligand recognition can be affected by point mutations outside of the binding pocket, which has implications for both the assignment and reengineering of riboswitches in this structural class.
Ren, A., Wang, X. C., Kellenberger, C. A., Rajashankar, K. R., Jones, R., Hammond, M. C. and Patel, D. J. (2015). Structural basis for molecular discrimination by a 3’,3’-cGAMP riboswitch. Cell Reports 11, 1-12.
Global RNA Fold and Molecular Recognition for a pfI Riboswitch Bound to ZMP, a Master Regulator of One Carbon Metabolism
ZTP, the pyrophosphorylated analog of ZMP (5-amino-4-imidazole carboxamide ribose-5’-monophpsphate), was identified as an alarmone that senses 10-formyl-tetrahydroflate deficiency in bacteria. Recently, a pfI riboswitch was identified that selectively binds ZMP and regulates genes associated with purine biosynthesis and one-carbon metabolism. We report on the structure of the ZMP-bound T. carboxydivorans pfI riboswitch sensing domain, thereby defining the pseudoknot-based tertiary RNA fold, the binding pocket architecture and principles underlying ligand recognition specificity. Molecular recognition involves shape complementarity, with the ZMP 5-amino and carboxamide groups paired with the Watson-Crick edge of an invariant uracil, the imidazole ring sandwiched between guanines, while the sugar hydroxyls form intermolecular hydrogen bond contacts. The burial of the ZMP base and ribose moieties, together with unanticipated coordination of the carboxamide by Mg2+, contrasts with exposure of the 5’-phosphate to solvent. Our studies highlight the principles underlying RNA-based recognition of ZMP, a master regulator of one-carbon metabolism.
Ren, A., Rajashankar, K. R. and Patel, D. J. (2015). Global fold and molecular recognition for the pfI riboswitch bound to ZMP, a master regulator of one carbon metabolism. Structure 23, 1375-1381.
The ydaO riboswitch, involved in sporulation, osmotic stress responses and cell wall metabolism, targets the second messenger c-di-AMP with subnanomolar affinity. We have solved the structure of c-di-AMP bound to the Thermoanaerobacter tengcongenesis ydaO riboswitch, thereby identifying a five-helical scaffold containing a zippered-up bubble, a pseudoknot and long-range tertiary base pairs. Highlights include the identification of two c-di-AMP binding pockets on the same face of the riboswitch, related by pseudo two-fold symmetry, with potential for cross-talk between sites mediated by adjacently-aligned base stacking alignments connecting pockets. The adenine rings of bound c-di-AMP molecules are wedged between bases and stabilized by stacking, base-sugar and sugar-sugar intermolecular hydrogen bonding interactions. The structural studies are complemented by ITC-based binding studies of mutants mediating key tertiary intermolecular contacts. The T. tengcongenesis ydaO riboswitch, like its B. subtilis counterpart, likely functions through a transcription termination mechanism, with the c-di-AMP bound state representing an ‘off’ switch.
Ren, A. and Patel, D. J. (2014). c-di-AMP binds the ydaO riboswitch in two pseudo-symmetry-related pockets. Nat. Chem. Biol. 10, 780-786.
Significant advances in our understanding of RNA architecture, folding and recognition have emerged from structure-function studies on riboswicthes, non-coding RNAs whose sensing domains bind small ligands and whose adjacent expression platforms contain RNA elements involved in the control of gene regulation. We now report on the ligand-bound structure of the Thermotoga petrophila fluoride riboswitch, which adopts a higher-order RNA architecture stabilized by pseudoknot and long-range reversed Watson-Crick and Hoogsteen A•U pair formation. The bound fluoride ion is encapsulated within the junctional architecture, anchored in place through direct coordination to three Mg2+ ions, which in turn are octahedrally coordinated to waters and five inwardly-pointing backbone phosphates. Our structure of the fluoride riboswitch in the bound state defines how RNA can form a binding pocket selective for fluoride, while discriminating against larger halide ions. The T. petrophila fluoride riboswitch most likely functions in gene regulation through a transcription termination mechanism.
Ren, A., Rajashankar, K. & Patel, D. J. (2012). Fluoride ion encapsulation by Mg2+ and phosphates in a fluoride riboswitch. Nature 486, 85-89.
Purine riboswitches play an essential role in genetic regulation of bacterial metabolism. This family includes the 2′-deoxyguanosine (dG) riboswitch, involved in feedback control of deoxyguanosine biosynthesis. To understand the principles that define dG selectivity, we determined crystal structures of natural Mesoplasma florum riboswitch bound to cognate dG, as well as non-cognate guanosine, deoxyguanosine monophophate and guanosine monophosphate. Comparison with related purine riboswitch structures reveals that the dG riboswitch achieves its specificity by modifying key interactions involving the nucleobase and through rearrangement of the ligand-binding pocket, so as to accommodate the additional sugar moiety. In addition, we observe novel conformational changes beyond the junctional binding pocket, extending as far as peripheral loop-loop interactions. It appears that re-engineering riboswitch scaffolds will require consideration of selectivity features dispersed throughout the riboswitch tertiary fold, and that structure-guided drug design efforts targeted to junctional RNA scaffolds need to be addressed within such an expanded framework.
Pikovskaya, O., Polonskkaya, A., Patel, D. J. & Serganov, A. (2011). Structural principles of nucleoside selectivity in a 2’-deoxyguanosine. Nat. Chem. Biol. 7, 748-755.
Long-range Pseudoknot Interactions Dictate the Regulatory Response in the Tetrahydrofolate Riboswitch
Tetrahydrofolate (THF), a biologically active form of the vitamin folate (B9), is an essential cofactor in one-carbon transfer reactions. In bacteria, expression of folate related genes is controlled by feedback modulation in response to specific binding of THF and related compounds to a riboswitch. Here, we present the x-ray structures of the THF-sensing domain from the Eubacterium siraeum riboswitch in the ligand-bound and unbound states. The structure reveals an ‘inverted’ three-way junctional architecture, most unusual for riboswitches, with the junction located far from the regulatory helix P1 and not directly participating in helix P1 formation. Instead, the three-way junction, stabilized by binding to the ligand, aligns the riboswitch stems for long-range tertiary pseudoknot interactions that contribute to the organization of helix P1 and therefore stipulate the regulatory response of the riboswitch. The pterin moiety of the ligand docks in a semi-open pocket adjacent to the junction, where it forms specific hydrogen bonds with two moderately conserved pyrimidines. The aminobenzoate moiety stacks on a guanine base, while the glutamate moiety does not appear to make strong interactions with the RNA. In contrast to other riboswitches, these findings demonstrate that the THF riboswitch uses a limited number of available determinants for ligand recognition. Given that modern antibiotics target folate metabolism, the THF riboswitch structure provide insights on mechanistic aspects of riboswitch function and may help in manipulating THF levels in pathogenic bacteria.
Huang, L., Ishibe-Murakami, S., Patel, D. J. & Serganov, A. (2011). Long-range pseudoknot interactions dictate the regulatory response in the tetrahydrofolate riboswitch. Proc. Natl. Acad. Scis. USA. 108, 14801-14806.
Glycine riboswitches regulate gene expression by feedback modulation in response to cooperative binding to glycine. Here, we report on crystal structures of the second glycine-sensing domain from the V. cholerae riboswitch in the ligand-bound and unbound states. This domain adopts a three-helical fold that centers on a three-way junction and accommodates glycine within a bulge-containing binding pocket above the junction. Glycine recognition is governed by specific interactions, assisted by Mg2+ cations, and shape complementarity with the pocket. A conserved adenine extrudes from the binding pocket and intercalates into the junction implying that glycine binding in the context of the complete riboswitch could impact on gene expression by stabilizing the riboswitch junction and regulatory P1 helix. Analysis of riboswitch interactions in the crystal and footprinting experiments indicate that adjacent glycine-sensing modules of the riboswitch could form specific interdomain interactions, thereby potentially contributing to the cooperative response.
Huang, L., Serganov, A. & Patel, D. J. (2010). Structural insights into ligand recognition by a sensing domain of the cooperative glycine riboswitch. Mol Cell 40, 774-786.
Many enzymatic reactions catalyzed by proteins require the use of coenzymes. Recently, the biosynthesis of several coenzymes has been demonstrated to be subject to feedback regulation by riboswitches. Flavin mononucleotide (FMN)-specific riboswitches, also known as RFN elements, direct expression of bacterial genes involved in the biosynthesis and transport of riboflavin (vitamin B2) and related compounds. We present here the crystal structures of the highly conserved metabolite-sensing domain of the Fusobacterium nucleatum riboswitch bound to FMN, riboflavin, and antibiotic roseoflavin. The FMN riboswitch structure, centered on an FMN-bound six-stem junction, does not fold by collinear stacking of adjacent helices, typical for folding of large RNAs. Rather, it adopts a butterfly-like scaffold, stapled together by opposingly directed but nearly identically folded peripheral domains. The pseudo-symmetric FMN moiety is positioned asymmetrically within the junctional site and is specifically bound to RNA through hydrogen-bonding and stacking of the isoalloxazine ring chromophore and through direct and Mg2+-mediated contacts with the terminal phosphate moiety. Our structural data, complemented by binding and footprinting experiments, are consistent with a largely pre-folded tertiary RNA architecture and identify conformational transitions within the junctional-binding pocket on FMN recognition. The inherent plasticity of the FMN-binding pocket and the availability of large openings next to the chromophore- and phosphate-binding sites make the riboswitch an attractive target for structure-based design of FMN-like antimicrobial compounds. In addition, our studies have explained the effects of spontaneous and antibiotic-induced deregulatory mutations and provided molecular insights into gene expression control by FMN riboswitches, features that are essential for understanding the FMN-based regulatory circuits in normal and riboflavin-overproducing bacterial strains.
Serganov, A., Huang, L. & Patel, D. J. (2009). Coenzyme recognition and gene regulation by a FMN riboswitch. Nature 458, 233-237. [PubMed Abstract]
In bacteria, the intracellular concentration of several amino acids is controlled by riboswitches. One of the important regulatory circuits involves lysine-specific riboswitches, which direct biosynthesis and transport of lysine and precursors common for lysine and other amino acids. To understand the molecular basis of amino acid recognition by riboswitches, we have determined the 1.9 Å crystal structure of the 174-nt sensing domain of the Thermatoga maritima lysine riboswitch in free and bound states. An independent structure of the Lysine riboswitch has been solved by the Robert Batey laboratory. The riboswitch features an unusual and intricate architecture, involving three-helical and two-helical bundles connected by a compact five-helical junction and stabilized by various long-range tertiary interactions. Lysine interacts with the junctional core of the riboswitch and is specifically recognized through shape-complementarity within the elongated binding pocket and through multiple direct and K+-mediated hydrogen bonds to its charged ends. Our structural and biochemical studies suggest pre-formation of the riboswitch scaffold and identify conformational changes associated with the formation of a stable lysine-bound state that prevents alternative folding of the riboswitch and facilitates formation of downstream regulatory elements. We have also determined several structures of the riboswitch bound to different lysine analogs, including antibiotics, in an effort to elucidate the ligand-binding capabilities of the lysine riboswitch and understand the nature of antibiotic resistance. Our results provide insights into a mechanism of lysine riboswitch-dependent gene control at the molecular level, thereby contributing to ongoing efforts at exploration of the pharmaceutical and biotechnological potential of riboswitches.
Serganov, A., Huang, L. & Patel, D. J. (2008). Structural insights into amino acid binding and gene control by a lysine riboswitch. Nature 455, 1263-1267. [PubMed Abstract]
We have solved the 2.05 crystal structure of the sensing domain of thiM mRNA from E. coli that responds to the coenzyme thiamine pyrophosphate (TPP), an active form of vitamin B1. The structure of TPP riboswitch from Arabidopsis thalinia has also been solved by the Nenad Ban laboratory at ETH-Zurich. The crystal structure reveals a complex-folded RNA scaffold wherein one domain forms a narrow pocket for the 4-amino-5-hydroxymethyl-2-methylpyrimidine (HMP) moiety of TPP, while another subdomain forms a wide pocket that uses divalent metal ions and water molecules to make bridging contacts between the sensing domain and the pyrophosphate moiety of TPP. The thiazole moiety of TPP is not recognized by the riboswitch, and this observation explains why the antibacterial compound pyrithiamine pyrophosphate can target this class of riboswitches. These findings also reveal how riboswitches can form precision binding pockets that rival those formed by protein genetic factors.
Serganov, A., Polonskaia, A., Phan, A. T., Breaker, R. R. & Patel, D. J. (2006). Structural basis for gene regulation by a riboswitch that senses thiamine pyrophosphate. Nature 411, 1167-1171. [PubMed Abstract]
We have solved the crystal structure of guanine- and adenine-sensing riboswitches, which together with the structure of the hypoxanthine-sensing riboswitch from the Robert Batey laboratory in Boulder, Colorado, provide molecular explanations for the exquisite discriminatory sensitivity to distinguish between bound guanine and adenine, associated with the metabolite encapsulation process. The riboswitches form tuning-fork-like architectures, in which the prongs are held in parallel through hairpin loop interactions, and the internal bubble zippers up to form the purine-binding pocket. The bound purines are held through hydrogen-bonding interactions involving conserved nucleotides along their entire periphery. Recognition specificity is associated with Watson-Crick pairing of the encapsulated adenine and guanine ligands with uridine and cytidine, respectively.
Serganov, A., Yuan, Y-R., Pikovskaya, O., Polonskaia, A., Malinina, L., Phan, A. T., Hobartner, C., Micura, R., Breaker, R. R. & Patel, D. J. (2004). Structural basis for discriminative regulation of gene expression by adenine- and guanine-sensing mRNAs. Chem. Biol. 11, 1729-1741. [PubMed Abstract]
The majority of efforts addressing the catalytic function of RNA have focused on natural and in vitro selected ribozymes and their divalent cation-modulated catalytic functionalities. Much less effort has been directed to RNA’s ability to catalyze chemical reactions, and the identification of RNA-based scaffolds that facilitate such processes.
The field of small self-cleaving nucleolytic ribozymes has been invigorated by the recent discovery of the twister, twister-sister, pistol and hatchet ribozymes. We report on the crystal structure of the env25 pistol ribozyme, which adopts a compact tertiary architecture stabilized by an embedded pseudoknot fold. The G-U cleavage site adopts a splayed-apart conformation with in-line alignment of the modeled 2’-O of G for attack on the adjacent to-be-cleaved P-O5’ bond. Highly conserved residues G40 (N1 position) and A32 (N3 and 2’-OH positions) are aligned to act as general base and general acid respectively to accelerate cleavage chemistry, with their roles confirmed from cleavage assays on mutants, and an increased pKa of 4.7 for A32. Our structure of the pistol ribozyme defines how the overall and local topologies dictate the in-line alignment at the G-U cleavage site, with cleavage assays on mutants identifying key residues participating in acid-base catalyzed cleavage chemistry.
Ren, A., Vusurovic, N., Gebetsberger, J., Gao, P., Juen M., Kreutz, C., Micura, R. & Patel, D. J. (2016). Pistol ribozyme adopts an embedded pseudoknot fold facilitating site-specific in-line self-cleavage. Nat. Chem. Biol. 12, 702-708.
Small self-cleaving nucleolytic ribozymes contain catalytic domains that accelerate site-specific cleavage/ligation of phosphodiester backbones. As part of a collaborative effort with the Ronald Micura laboratory (University of Innsbruck, Austria), we reported on the 2.9-Å crystal structure of the env22 twister ribozyme, which adopts a compact tertiary fold stabilized by co-helical stacking, double-pseudoknot formation and long-range pairing interactions. The U-A cleavage site adopts a splayed-apart conformation with the modeled 2’-O of U positioned for in-line attack on the adjacent to-be-cleaved P-O5’ bond. Both an invariant guanosine and a Mg2+ are directly coordinated to the non-bridging phosphate oxygens at the U-A cleavage step, with the former positioned to contribute to catalysis and the latter to structural integrity. The impact of key mutations on cleavage activity identified an invariant guanosine that contributes to catalysis. Our structure of the in-line aligned env22 twister ribozyme is compared with two recently-reported off-line orthogonally-aligned structures of twister ribozymes, which adopt similar global folds, but differ in conformational features around the cleavage site.
Ren, A., Kosutic, M., Rajashankar, K. R., Frener, M., Santner, T., Westhof, E., Micura, R. and Patel, D. J. (2014). In-line alignment and Mg2+ coordination at the cleavage site of the twister ribozyme. Nat. Commun. 15: 5534.
Kosutic, M., Neuner, S., Ren, A., Flur, S., Wunderlich, C., Mayrhofer, E., Vusurovic, N., Seikowski, J., Westhof, E., Hobartner, C., Patel, D. J., Kreitz, C. and Micure, R. (2015). A mini-twister variant and impact of residues/cations on the phosphodiester cleavage chemistry of this ribozyme class. Angew. Chemie Int. Edn. 54, 15128-15133.
An extremely interesting system for structural and mechanistic characterization is the Diels-Alder ribozyme identified in our collaborator Andres Jaschke’s laboratory at the University of Heildelberg, Germany, because of its moderate size, 20,000-fold rate enhancement, and the documented enantiomeric selectivity associated with the catalyzed cyclization reaction. The catalytic binding pocket needs to accommodate ligands of different sizes and shapes as the reaction proceeds from reactants to transition state intermediates to products. We have solved the crystal structure of the Diels-Alder ribozyme, which catalyzes the cyclization of anthracene and N-pentyl maleimide, in the unbound state and in complex with the reaction product. The RNA adopts a λ-shaped nested pseudoknot architecture whose preformed hydrophobic pocket is precisely complementary in shape to the reaction product. RNA folding and product binding are dictated by extensive stacking and hydrogen bonding, whereas stereoselection is governed by the shape of the catalytic pocket. Catalysis is apparently achieved by a combination of proximity, steric complementarity, and electronic effects. We have observed structural parallels in the independently evolved catalytic pocket architectures of ribozyme- and antibody-catalyzed Diels-Alder carbon-carbon bond-forming reactions. The recognition principles identified for the Diels-Alder ribozyme in the free and product-bound states should provide a platform for the design of engineered catalysts with tailored specificities and selectivities.
Serganov, A., Keiper, S., Malinina, L., Tereschko, V., Skripkin, E., Hobartner, C., Polonskaia, A., Phan, A. T., Wombacher, R., Micura, R., Dauter, Z., Jaschke, A. & Patel, D. J. (2005). Structural basis for Diels-Alder ribozyme catalyzed carbon-carbon bond formation. Nature Struct. Mol. Biol. 12, 218-224. [PubMed Abstract]