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 discovered in the laboratory of our collaborator Ronald Breaker laboratory of Yale University, given the new and unexpected role for mRNA 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 review on riboswitches and ribozymes:
Serganov, A. & Patel, D. J. (2007). Ribozymes, riboswitches and beyond: regulation of gene expression without proteins. Nat. Rev. Genetics 8, 776-790. [PubMed Abstract]
Flavin Mononucleotide Riboswitch
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]
The structural efforts are currently being extended to other metabolite-sensing riboswitches discovered in the Ronald Breaker laboratory. We anticipate that a structural database of principles related to recognition of the aptamer scaffolds of metabolite-sensing mRNAs could underlie new approaches to drug design and to development of molecular sensors.
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]
Thiamine Pyrophosphate Riboswitch
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.
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]
In the future, we shall continue our efforts to biochemically and structurally characterize additional novel ribozymes catalyzing distinct chemical reactions, with controllable catalytic activities, tunable specificities, and enantiomeric capabilities.