Abstract

The discovery that RNA can catalyze reactions has changed our view of the role of RNA in biological systems. There are presently a large number of systems for which RNA has been shown to be the biological catalyst, and the hammerhead RNA cleaving domain is one of the best studied systems. Although there is a great deal of information on the biochemical and kinetic properties of the hammerhead domain, there is presently no direct structural data on this system. Thus we will use nuclear magnetic resonance (NMR) spectroscopy to generate structural data on the hammerhead self-cleaving RNA domain and also on a related less well-characterized system, the hairpin self-cleaving RNA domain. We will primarily concentrate on determining the tertiary structure, the structure of the active site, and structure-activity relations in both systems. The hammerhead and hairpin ribozymes have site-specific RNA endonuclease activities and have been shown in vitro to efficiently and catalytically cleave specific sites in target RNAS. These results suggest important in vivo applications where one could design a ribozyme to specifically cleave a target mRNA or the genome of an RNA virus such as HIV. The direct structural information from our NMR studies combined with the kinetic, mechanistic and biochemical properties of these ribozymes will aid in the design of optimal RNA cleavage reagents.

  Funding Agency

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI030726-02
Application #
3145769
Study Section
Molecular and Cellular Biophysics Study Section (BBCA)
Project Start
1990-12-01
Project End
1993-11-30
Budget Start
1991-12-01
Budget End
1992-11-30
Support Year
2
Fiscal Year
1992
Total Cost
Indirect Cost

  Institution

Name
University of Colorado at Boulder
Department
Type
Schools of Arts and Sciences
DUNS #
City
Boulder
State
CO
Country
United States
Zip Code
80309

  Publications

Boots, Jennifer L; Canny, Marella D; Azimi, Ehsan et al. (2008) Metal ion specificities for folding and cleavage activity in the Schistosoma hammerhead ribozyme. RNA 14:2212-22
Canny, Marella D; Jucker, Fiona M; Pardi, Arthur (2007) Efficient ligation of the Schistosoma hammerhead ribozyme. Biochemistry 46:3826-34
Lee, Joon-Hwa; Pardi, Arthur (2007) Thermodynamics and kinetics for base-pair opening in the P1 duplex of the Tetrahymena group I ribozyme. Nucleic Acids Res 35:2965-74
Latham, Michael P; Brown, Darin J; McCallum, Scott A et al. (2005) NMR methods for studying the structure and dynamics of RNA. Chembiochem 6:1492-505
Canny, Marella D; Jucker, Fiona M; Kellogg, Elizabeth et al. (2004) Fast cleavage kinetics of a natural hammerhead ribozyme. J Am Chem Soc 126:10848-9
Jucker, Fiona M; Phillips, Rebecca M; McCallum, Scott A et al. (2003) Role of a heterogeneous free state in the formation of a specific RNA-theophylline complex. Biochemistry 42:2560-7
Bondensgaard, Kent; Mollova, Emilia T; Pardi, Arthur (2002) The global conformation of the hammerhead ribozyme determined using residual dipolar couplings. Biochemistry 41:11532-42
Sibille, N; Pardi, A; Simorre, J P et al. (2001) Refinement of local and long-range structural order in theophylline-binding RNA using (13)C-(1)H residual dipolar couplings and restrained molecular dynamics. J Am Chem Soc 123:12135-46
Zimmermann, G R; Wick, C L; Shields, T P et al. (2000) Molecular interactions and metal binding in the theophylline-binding core of an RNA aptamer. RNA 6:659-67
Hansen, M R; Simorre, J P; Hanson, P et al. (1999) Identification and characterization of a novel high affinity metal-binding site in the hammerhead ribozyme. RNA 5:1099-104

Showing the most recent 10 out of 19 publications