Research Interests:
Our group is broadly interested in the chemistry and biochemistry of nucleic acids, with particular emphasis on RNA and RNA catalysis. Our laboratory integrates organic chemistry, physical chemistry, enzymology, and molecular biology to gain a fundamental understandingof nucleic acid structure and mechanisms of RNA catalysis. Using the principles and techniques of organic chemistry and molecular biology, we manipulate the structure of RNA molecules at precise locations in ways that are designed to elucidate questions about biological function.
Mechanism of RNA Catalysis
We aim to gain a fundamental understanding of the role divalent metal ions play in phosphoryl transfer reactions that occur during RNA splicing, a fundamental step in genetic expression. One experimental system that we are using to address these issues is the self-splicing intervening sequence RNA of the ciliated protozoan Tetrahymena. Shortened forms of this RNA can act as enzymes, catalyzing the sequence-specific cleavage of RNA and DNA substrates with multiple turnover. We use sulfur substitution of oxygen substituents on the phosphoryl group undergoing transfer to reveal the transition state interactions between the ribozyme and the scissile phosphate. We are also interested in the development of new methods and model systems for studying RNA molecules. For example, we recently designed a series of nucleoside analogues in which the C2Õ-beta hydrogen atom ofthe ribose is replaced by CH3, CH2F, CHF2, or CF3. These analogues provide a systematic way to perturb the acidity of the 2'-OH group, thereby allowing us to probe the important role of this functional group in RNA-mediated biological processes.
RNA-Protein Interactions
Restrictocin is a small protein (149 amino acids) so toxic that a single molecule can kill an entire cell. This protein, from Aspergillus restrictus, is a member of a group of functionally homologous cytotoxins that includes the better-known sarcin. Its mechanism of toxicity is fascinating: a single protein can cross the cell membrane and cleave the 23Ð28S ribosomal RNA at a single phosphodiester bond. The cleavage site resides within a region of the ribosomal RNA known as the sarcin/ricinloop (SRL), which folds into a tetraloop motif and abulged-G motif. The SRL participates in the binding of elongation factors during protein synthesis. Considering that the 28S ribosomal RNA contains thousands of phosphodiester bonds, the apparent specificity of this ribonuclease is remarkable. This single cleavage event inactivates the ribosome and consequently abolishes its ability to carry out protein synthesis, which ultimately leads to death of the cell.
This scenario immediately prompts a number of questions: How does the protein cross the cell membrane? Does it really possess the attributed specificity? Is every ribosome in the cell inactivated or does a single inactivation event lead to activation of an apoptotic pathway?Additionally, the potency of this protein immediately suggests a potential clinic use as an anticancer drug. All of these are interesting questions that we hope to answer. In addition, this system has broader significance in biology as a model system to study RNA-protein interactions, which are ubiquitous and mediate numerous important events during gene expression. The crystal structures of restrictocin and the SRL RNA have been solved in isolation, and Carl Correll's lab (University of Chicago) has solved a structure of an SRL analog in complex with restrictocin. Upon complex formation the geometry of the tetraloop is dramatically rearranged by base restacking and base flipping. Remarkably, few functional studies have been reported on this protein. Our initial focus will be to determine the dynamic changes that occur in the SRL when it binds to restrictocin and to elucidate the energetic contributions that enzyme-RNA substrate contacts play in cleavage-site recognition and catalysis.
Selected References
Hougland, J.L., Kravchuk, A.V., Herschlag, D. & Piccirilli, J.A. Functional identification of catalytic metal ion binding sites within RNA. PLoS Biology 3, 1536-1548 (2005).
Das, S.R. & Piccirilli, J.A. General acid catalysis by the hepatitis delta virus ribozyme. Nat Chem Biol 1, 45-52 (2005).
Korennykh, A.V., Piccirilli, J.A. & Correll, C.C. The electrostatic character of the ribosomal surface enables extraordinarily rapid target location by ribotoxins. Nat Struct Biol 13, 436-443 (2006).
Gordon, P.M., Fong, R. & Piccirilli, J.A. A Second Divalent Metal Ion in the Group II Intron Reaction Center.Chem Biol 14, 607-612 (2007).
Ye, J.D., Li, N.S., Dai, Q. & Piccirilli, J.A. The mechanism of RNA strand scission: an experimental measure of the Bronsted coefficient, beta nuc. Angew Chem Int Ed Engl 46, 3714-3717 (2007).
Ye, J.D. et al. Synthetic antibodies for specific recognition and crystallization of structured RNA. Proc Natl Acad Sci USA 105, 82-87 (2008).
Hougland, J.L., Sengupta, R.N., Dai, Q., Deb, S.K. & Piccirilli, J.A. The 2'-hydroxyl group of the guanosine nucleophile donates a functionally important hydrogen bond in the tetrahymena ribozyme reaction.Biochemistry 47, 7684-7694 (2008).
Plantinga, M.J., Korennykh, A.V., Piccirilli, J.A. & Correll, C.C. Electrostatic interactions guide the active site face of a structure-specific ribonuclease to its RNA substrate. Biochemistry 47, 8912-8918 (2008). PMID: 18672906 [PubMed - as supplied by publisher]
Dai, Q., Saikia, M., Li, N-S., Pan, T., and Piccirilli, J.A. Efficient chemical synthesis of AppDNA by adenylation of immobilized DNA-5'-monophosphate. Org. Lett., 11, 1067-1070 (2009). PMID: 19191584 [PubMed - as supplied by publisher].
Forconi, M., Sengupta, R.N., Piccirilli, J.A. & Herschlag, D. Structure and function converge to identify a hydrogen-bond in the Group I ribozyme active site. Angew Chem Int Ed Engl, in press (2009).