An integrated series of biochemical, biophysical, and combinatorial experiments is designed to answer three fundamental questions concerning the hairpin ribozyme: First, how does the RNA assemble into a catalytic structure? Second, what is the structure of the ribozyme-substrate complex? Third, how does this structure result in the acquisition of catalytic activity? The hairpin ribozyme is well suited to such experiments, since the molecule is small enough that comprehensive studies can be undertaken, yet complex enough that significant conformational changes occur during the catalytic cycle. The hairpin differs in several important respects from the only two RNA enzymes that have been more extensively studied, the hammerhead ribozyme and group I introns.
Specific Aims are: (1) Map the tertiary structure of the ribozyme-substrate complex; (2) Determine the mechanisms through which metal ions and inhibitors modulate catalytic activity; (3) Define the folding pathway and determine the requirements, kinetics, and thermodynamics of folding and unfolding; (4) Determine the tertiary structure of the ribozyme- substrate complex at high resolution; and (5) Integrate results to develop and test models of the catalytic mechanism. The importance of this work includes advancement of our understanding of RNA structure and how it leads to catalytic activity, the role of catalytic RNA and RNP in biological reactions, and the application of our understanding of catalytic RNA and ribozyme engineering to improving heath, through identifying gene function in genomic investigations and the development of selective ribozyme-based therapeutics for genetic and viral diseases.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI044186-04
Application #
6475524
Study Section
Biochemistry Study Section (BIO)
Program Officer
Tseng, Christopher K
Project Start
1998-12-01
Project End
2003-11-30
Budget Start
2001-12-01
Budget End
2002-11-30
Support Year
4
Fiscal Year
2002
Total Cost
$439,331
Indirect Cost
Name
University of Vermont & St Agric College
Department
Microbiology/Immun/Virology
Type
Schools of Medicine
DUNS #
066811191
City
Burlington
State
VT
Country
United States
Zip Code
05405
Kraemer-Chant, Christina M; Heckman, Joyce E; Lambert, Dominic et al. (2014) Cobalt(III)hexaammine-dependent photocrosslinks in the hairpin ribozyme. J Inorg Biochem 131:87-98
Buskiewicz, Iwona A; Burke, John M (2012) Folding of the hammerhead ribozyme: pyrrolo-cytosine fluorescence separates core folding from global folding and reveals a pH-dependent conformational change. RNA 18:434-48
Gaur, Snigdha; Heckman, Joyce E; Burke, John M (2008) Mutational inhibition of ligation in the hairpin ribozyme: substitutions of conserved nucleobases A9 and A10 destabilize tertiary structure and selectively promote cleavage. RNA 14:55-65
Lambert, Dominic; Heckman, Joyce E; Burke, John M (2006) Cation-specific structural accommodation within a catalytic RNA. Biochemistry 45:829-38
Pinard, Robert; Lambert, Dominic; Pothiawala, Gulnar et al. (2004) Modifications and deletions of helices within the hairpin ribozyme-substrate complex: an active ribozyme lacking helix 1. RNA 10:395-402
Hampel, K J; Burke, J M (2001) A conformational change in the ""loop E-like"" motif of the hairpin ribozyme is coincidental with domain docking and is essential for catalysis. Biochemistry 40:3723-9
Pinard, R; Lambert, D; Heckman, J E et al. (2001) The hairpin ribozyme substrate binding-domain: a highly constrained D-shaped conformation. J Mol Biol 307:51-65
Pinard, R; Hampel, K J; Heckman, J E et al. (2001) Functional involvement of G8 in the hairpin ribozyme cleavage mechanism. EMBO J 20:6434-42
Pinard, R; Lambert, D; Walter, N G et al. (1999) Structural basis for the guanosine requirement of the hairpin ribozyme. Biochemistry 38:16035-9