 |
| 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, Associate 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. |
|
|
| Joseph
A. Piccirilli |
| Associate
Professor |
|
|
| |
| Research
Interests: |
| 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. |
| |
| RNA-Protein
Interactions |
| 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. |
| |
| 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 U S A 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). |
| |
|
Organic Chemistry 