Gene Regulation by Riboswitches and Ribozymes

Gene Regulation by Riboswitches and Ribozymes


The role of RNA in information transfer and catalysis highlights its dual functionalities. Our laboratory has a long-standing interest in RNA folding, recognition, and catalysis. We are especially interested in both natural and in vitro selected RNA aptamer-based systems, because they serve as exceptional scaffolds for ligand recognition and catalysis, exhibiting tunable specificities and enantiomeric selectivities.


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 RNAs as riboswitches, and because structural-energetics information will be critical for defining allosteric mRNA transitions associated with the modulation of gene expression levels and metabolic homeostasis.

We have written two reviews on riboswitches (Serganov & Patel, 2007, 2012).

Serganov, A. & Patel, D. J. (2007). Ribozymes, riboswitches and beyond: regulation of gene expression without proteins. Nat. Rev. Genetics 8, 776-790.

Serganov, A. & Patel, D. J. (2012). Metabolite recognition principles and molecular mechanisms underlying riboswitch function. Ann. Rev. Biophys. 41, 343-370.


Nucleoside and Cyclic Nucleoside Ligands. In collaboration with the Ronald Micura lab (University of Innsbruck), we have solved the crystal structure of guanine- and adenine-sensing riboswitches, which provide molecular explanations for the exquisite discriminatory sensitivity to distinguish between bound guanine and adenine, associated with the metabolite encapsulation process (Serganov et al. 2004). The purine-sensing 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. Recognition specificity is associated with Watson-Crick pairing of the encapsulated adenine and guanine ligands with uridine and cytidine, respectively. Notably, the deoxy guanosine 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 (Pikovskaya et al. 2011).

We have solved the structure of c-di-AMP bound to the T. tengcongenesis ydaO riboswitch, thereby identifying a five-helical scaffold containing a zippered-up bubble, a pseudoknot and long-range tertiary base pairs (Ren & Patel, 2014). Highlights include the identification of two c-di-AMP binding pockets containing wedged adenine bases on the same face of the riboswitch, with potential for cross-talk between sites mediated by adjacently-aligned base stacking alignments connecting c-di-AMP pockets.  In a collaborative effort with the Ming Hammond lab (UCal-Berkeley), we solved structures of the aptamer domain of the 3’,3’-cGAMP riboswitch from Geobacter in the 3’,3’-cGAMP and c-di-GMP bound states (Ren et al. 2015). Structure-guided biochemical experiments revealed that specificity of ligand recognition can be affected by point mutations outside of the binding pocket, with implications for reengineering of this class of riboswitches.

Serganov, A. et al., 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.

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.

Ren, A. & Patel, D. J. (2014). c-di-AMP binds the ydaO riboswitch in two pseudo-symmetry-related pockets. Nat. Chem. Biol. 10, 780-786.

Ren, A. et al., Hammond, M. C. and Patel, D. J. (2015). Structural basis for molecular discrimination by a 3’,3’-cGAMP riboswitch. Cell Reports 11, 1-12.


Anion Ligands. Of particular interest is how RNA as a negatively charged polyphosphate can bind ligands that are also negatively charged. A riboswitch from P. syringae, which activates the expression of genes that encode putative fluoride transporters, has been identified that targets fluoride ion and discriminates against other halogen ions. We have solved the ligand-bound structure of the T. petrophila fluoride riboswitch, which adopts a higher-order RNA architecture stabilized by pseudoknot and long-range non-canonical pairs (Ren et al. 2012). 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.

Ren, A., Rajashankar, K. & Patel, D. J. (2012). Fluoride ion encapsulation by Mg2+ and phosphates in a fluoride riboswitch. Nature 486, 85-89.


Amino Acid Ligands. We have determined the crystal structure of the sensing domain of the T. maritima lysine riboswitch in free and bound states (Serganov et al. 2008). 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 through multiple direct and K+-mediated hydrogen bonds to its charged ends. We have also determined several structures of the riboswitch bound to different lysine analogs, including antibiotics, in an effort to elucidate the nature of antibiotic resistance.

We have solved the crystal structures of the second glycine-sensing domain from the V. cholerae riboswitch in the ligand-bound and unbound states (Huang et al. 2010). 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 a pair of hydrated Mg2+ cations, and shape complementarity with the pocket. Analysis of riboswitch interactions indicate that adjacent glycine-sensing modules of the riboswitch could form specific interdomain interactions, thereby potentially contributing to the cooperative response.

By combining X-ray, NMR and MD as part of a collaborative effort with the Hashim Al-Hashimi lab (Duke University), we characterized a L-glutamine-dependent conformational transition in the S. elongatus glutamine riboswitch from tuning-fork to L-shaped alignment of stem segments (Ren et al. 2015). This transition, involving formation of a long-range ‘linchpin’ Watson-Crick G-C pair capping interaction, generates an open ligand-binding pocket with L-glutamine selectivity enforced by Mg2+-mediated intermolecular interactions.

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.

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.

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.


Cofactor Ligands. We have solved the 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 (Serganov et al. 2006). The crystal structure reveals a complex-folded RNA scaffold with a pair of linked binding pockets, wherein one domain forms a narrow pocket for the aromatic 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.

We have solved the crystal structures of the metabolite-sensing domain of the F. nucleatum riboswitch bound to FMN, riboflavin, and antibiotic roseoflavin (Serganov et al. 2009). The structure of the FMN riboswitch, centered on an FMN-bound six-stem junction 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 anchors bound FMN through hydrogen-bonding and stacking of the isoalloxazine ring and through direct and Mg2+-mediated contacts with the terminal phosphate moiety.

We have solved the crystal structures of the THF-sensing domain from the E. siraeum riboswitch in the ligand-bound and unbound states (Serganov et al. 2011). The structure reveals an ‘inverted’ three-way junctional architecture, stabilized by binding to the ligand, thereby aligning 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.

A pfI riboswitch was identified that selectively binds ZMP and regulates genes associated with purine biosynthesis and one-carbon metabolism. We solved the structure of the ZMP-bound T. carboxydivorans pfI riboswitch sensing domain, thereby defining the pseudoknot-based tertiary RNA fold, with burial of the ZMP base and ribose moieties, together with unanticipated coordination of the carboxamide by Mg2+, which contrasts with exposure of the 5’-phosphate to solvent (Ren et al. 2015).

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.

Serganov, A., Huang, L. & Patel, D. J. (2009). Coenzyme recognition and gene regulation by a FMN riboswitch. Nature 458, 233-237.

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.

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.


Nucleolytic Ribozymes

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 self-cleavage processes with implications for viral genome replication, pre-mRNA processing, and alternative splicing. Small self-cleaving nucleolytic ribozymes contain catalytic domains that accelerate site-specific cleavage/ligation of phosphodiester backbones. The field of nucleolytic ribozymes has been invigorated by the recent discovery of the twister, twister-sister, pistol and hatchet nucleolytic ribozymes.

We have written a review on nucleolytic ribozymes in collaboration with Ronald Micura  (University of Innsbruck) (Ren et al. 2017).

Ren, A., Micura, R. & Patel, D. J. (2017). Structure-based mechanistic insights into catalysis by small self-cleaving ribozymes. Curr. Opin. Chem. Biol. 41, 71-83.


Twister and Twister-Sister Ribozymes. As part of a collaborative effort with the Ronald Micura lab (University of Innsbruck), we reported on the 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 (Ren et al. 2014). 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 (Kosutic et al. 2015).

The crystal structure and cleavage assays of a four-way junctional twister-sister self-cleaving ribozyme has been solved as part of a collaborative effort championed by the Ronald Micura and Aiming Ren (Zhejiang University) labs (Zheng et al. 2017). Notably, conserved spatially separated loop nucleotides are brought into close proximity at the ribozyme core through long-range interactions mediated by hydrated Mg2+ cations. The cytosine-adenosine step at the cleavage site adopts a splayed-apart orientation, with flexible cytosine directed outwards, whereas the adenosine is directed inwards and anchored by stacking and hydrogen-bonding interactions. Structure-guided mutational studies suggest contributions to the cleavage chemistry from interactions between a guanine at the active site and the non-bridging oxygen of the scissile phosphate, a feature found previously also for the related twister ribozyme.

Kosutic, M. et al., 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.

Ren, A. et al.., 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.

Zheng, L. et al., Patel, D. J., Micura, R. & Ren, A. (2017). Structure-based insights into self-cleavage by a four-way junctional twister-sister ribozyme. Nat. Commun. 8:1180.


Pistol Ribozyme. In collaboration with the Ronald Micura lab, we report on the crystal structure of the env25 pistol ribozyme, which adopts a compact tertiary architecture stabilized by an embedded pseudoknot fold (Ren et al. 2016). 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 guanine and adenine residues 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 the adenine (Neuner et al. 2017). We have also identified a second hydrated Mg2+ ion that approaches the scissile phosphate from its binding site in the pre-cleavage state to reach out for water-mediated hydrogen bonding in the cyclophosphate product (Teplova et al. 2020).

Neuner, S. et al., Ren, A., Patel, D. J. & Micura, R. (2017) Atom-specific mutagenesis of an active site-adenine in the pistol ribozyme reveals its structural rather than catalytic roie. Angew. Chemie. Int. Edn 56, 15954-15958.

Ren, A. et al., Micura, R. & Patel, D. J. (2016). Pistol ribozyme adopts an embedded pseudoknot fold facilitating site-specific in-line self-cleavage. Nat. Chem. Biol. 12, 702-70

Teplova, M. et al., Ren, A., Patel, D. J. & Micura R. (2020). On crucial roles of two hydrated Mg2+ ions in reaction catalysis of the pistol ribozyme. Angew. Chemie. Int. Edn. 59, 2837-2843.


Hatchet Ribozyme. The Ronald Micura and Aiming Ren labs have championed the crystal structure determination of the hatchet ribozyme product, which adopts a compact pseudo-symmetric dimeric scaffold, with each monomer stabilized by long-range interactions involving highly conserved nucleotides brought into close proximity of the scissile phosphate. (Zheng et al. 2019) Strikingly, the catalytic pocket contains a cavity capable of accommodating both the modeled scissile phosphate and its flanking 5’ nucleoside. We identify a guanine lining the catalytic pocket positioned to contribute to cleavage chemistry.

Zheng, L. et al., Patel, D. J., Micura, R. & Ren, A. (2019). Hatchet ribozyme structure and implications for the precatalytic fold and cleavage mechanism. Proc. Natl. Acad. Scis. USA  116, 10783-10791.


Diels-Alder Ribozyme. An interesting system for structural and mechanistic characterization is the Diels-Alder ribozyme identified in our collaborator Andres Jaschke’s lab (University of Heildelberg), 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 both the unbound state and in complex with the reaction product (Serganov et al. 2005). The RNA adopts a lambda-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.

Serganov, A. et al. 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.