Riboswitches
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.
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, provides 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.
We have solved the 2.05 Å 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 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.
The structural efforts are currently being extended to other metabolite-sensing riboswitches discovered in the Ronald Breaker laboratory. We anticipate that a structural data-base 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.
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 such processes.
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An extremely interesting system for structural and mechanistic characterization is the Diels-Alder ribozyme identified in our collaborator Andres Jaschke's laboratory, 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  -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.
In the future, we shall continue to collaborate with the Andres Jaschke laboratory to identify and biochemically and structurally characterize novel ribozymes catalyzing distinct chemical reactions, with controllable catalytic activities, tunable specificities and enantiomeric capabilities.
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.
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