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| 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. |
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| Joseph A. Piccirilli |
| Associate Professor |
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| Research Interests: |
| Our group is broadly interested in the chemistry and biochemistry
of nucleic acids with particular emphasis on
RNA and RNA catalysis. The laboratory integrates areas
of organic chemistry, physical chemistry, enzymology
and molecular biology to gain a fundamental understanding
of 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 answer very specific questions
about biological function. |
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| Mechanism of RNA Catalysis |
| We employ these approaches toward gaining a fundamental
understanding of the role that 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 have used sulfur substitution
of the oxygen substituents on the phosphoryl group
undergoing transfer to reveal the transition state interactions
between the ribozyme and the scissile phosphate.
Another area of interest is the development of new methods
and model systems for studying RNA molecules. For
example, we have recently designed a series of nucleoside
analogues, in which the C2Õ-beta hydrogen atom of
the 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 all important role of this functional group in RNA
mediated biological processes. |
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| RNA-Protein Interactions |
| Restrictocin is a small protein (149 amino acids) that is
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, which includes
the better-known sarcin, and the mechanism of toxicity is
fascinating. The single protein is able to cross the cell
membrane and cleave the 23Ð28S ribosomal RNA at a
single phosphodiester bond. The cleavage site resides in
a region of the ribosomal RNA known as the sarcin/ricin
loop (SRL), which folds into a tetraloop motif and a
bulged-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. |
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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 CorrellÕs 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. |
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| Selected References |
| Ye, J. D., Tereshko, V., Frederiksen, J., Koide, A., Fellouse, F., Sidhu, S., Koide, S., Kossiakoff, T., and Piccirilli Joseph, A. Synthetic antibodies for specific recognition and crystallization of structured RNA. Proc Natl Acad Sci U S A, 105 82-87 (2008) |
| Korennykh, A. V., Plantinga, M. J., Correll, C. C., and Piccirilli, J. A. Linkage between Substrate Recognition and Catalysis during Cleavage of Sarcin/Ricin Loop RNA by Restrictocin. Biochemistry, 46 12744-12756 (2007) |
| Dai, Q., Fong, R., Saikia, M., Stephenson, D., Yu, Y. T., Pan, T., and Piccirilli, J. A. Identification of recognition residues for ligation-based detection and quantitation of pseudouridine and N6-methyladenosine. Nucleic Acids Res, 35 6322-6329 (2007) |
| Ye, J. D., Li, N. S., Dai, Q., and Piccirilli, J. A. The mechanism of RNA strand scission: an experimental measure of the Bronsted coefficient, beta nuc. Angew Chem Int Ed Engl, 46 3714-7 (2007). |
| Gordon, P. M., Fong, R., and Piccirilli, J. A. A Second Divalent Metal Ion in the Group II Intron Reaction Center. Chem Biol , 14 607-612 (2007). |
| Korennykh, A. V., Piccirilli, J. A., and Correll, C. C. The electrostatic character of the ribosomal surface enables extraordinarily rapid target location by ribotoxins. Nature Structural Biol., 13 436-443 (2006). |
| Das, S. R., and Piccirilli, J. A. General acid catalysis by the hepatitis delta virus ribozyme. Nat Chem Biol 1 45-52 (2005). |
| Hougland, J. L., Kravchuk, A. V., Herschlag, D., and Piccirilli, J. A. The ol'switcheroo shows how an RNA enzyme splices itself. PLoS Biology 3 1512 (2005). |
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Organic Chemistry 