Joseph A. Piccirilli Professor
Born Wilkes-Barre, Pennsylvania, 1960.
University of Scranton, B.Sc., 1982.
Rheinisch Westfalische Technische Hochschule Aachen, Fulbright Scholar, 1983.
Harvard University, Ph.D., 1989.
Harvard Traveling Scholar, Swiss Federal Institute of Technology, 1986-1989.
University of Colorado at Boulder, Howard Hughes Postdoctoral Research Fellow, 1989-1993.
The University of Chicago, Assistant Professor, 1993-2000.
Assistant Investigator, Howard Hughes Medical Institute, 1994-2000.
The University of Chicago, Professor, 2000-.
Associate Investigator, Howard Hughes Medical Institute, 2000-2004.
Investigator, Howard Hughes Medical Institute, 2004-.
Joint Appointment in the Department of Biochemistry and Molecular Biology.
OFFICE: Room CIS 406, Center for Integrative Science 929 East 57th Street, Chicago, IL 60637
Our group is broadly interested in the chemistry and biochemistryof nucleic acids with particular emphasis onRNA and RNA catalysis. The laboratory integrates areasof organic chemistry, physical chemistry, enzymologyand molecular biology to gain a fundamental understandingof nucleic acid structure and mechanisms ofRNA catalysis. Using the principles and techniques oforganic chemistry and molecular biology, we manipulatethe structure of RNA molecules at precise locations inways that are designed to answer very specific questionsabout biological function.
Mechanism of RNA Catalysis
We employ these approaches toward gaining a fundamentalunderstanding of the role that divalent metal ionsplay in phosphoryl transfer reactions that occur duringRNA splicing, a fundamental step in genetic expression.One experimental system that we are using to addressthese issues is the self-splicing intervening sequenceRNA of the ciliated protozoan Tetrahymena. Shortenedforms of this RNA can act as enzymes, catalyzing thesequence specific cleavage of RNA and DNA substrateswith multiple turnover. We have used sulfur substitutionof the oxygen substituents on the phosphoryl groupundergoing transfer to reveal the transition state interactionsbetween the ribozyme and the scissile phosphate.Another area of interest is the development of new methodsand model systems for studying RNA molecules. Forexample, we have recently designed a series of nucleosideanalogues, 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 theacidity of the 2'-OH group, thereby allowing us to probethe all important role of this functional group in RNAmediated biological processes.
Restrictocin is a small protein (149 amino acids) that isso toxic that a single molecule can kill an entire cell. Thisprotein from Aspergillus restrictus is a member of a groupof functionally homologous cytotoxins, which includesthe better-known sarcin, and the mechanism of toxicity isfascinating. The single protein is able to cross the cellmembrane and cleave the 23Ð28S ribosomal RNA at asingle phosphodiester bond. The cleavage site resides ina 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 ofelongation factors during protein synthesis. Consideringthat the 28S ribosomal RNA contains thousands of phosphodiesterbonds, the apparent specificity of this ribonucleaseis remarkable. This single cleavage event inactivatesthe ribosome and consequently abolishes its abilityto carry out protein synthesis, which ultimately leadsto 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 everyribosome in the cell inactivated or does a single inactivationevent lead to activation of an apoptotic pathway?Additionally, the potency of this protein immediatelysuggests a potential clinic use as an anticancer drug. Allof these are interesting questions that we hope to answer.In addition, this system has broader significance in biologyas a model system to study RNA-protein interactions,which are ubiquitous and mediate numerous importantevents during gene expression. The crystal structures ofrestrictocin and the SRL RNA have been solved in isolation,and Carl CorrellÕs lab (University of Chicago) hassolved a structure of an SRL analog in complex withrestrictocin. Upon complex formation the geometry ofthe tetraloop is dramatically rearranged by base restackingand base flipping. Remarkably, few functional studieshave been reported on this protein. Our initial focus willbe to determine the dynamic changes that occur in theSRL when it binds to restrictocin and to elucidate theenergetic contributions that enzyme-RNA substrate contactsplay in cleavage-site recognition and catalysis.
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).