To achieve its diverse range of biological functions, RNA must fold into specific tertiary structures that create active sites for chemical transformations or recognition features for protein binding. Conformational transitions necessary to achieve biological activity involve large movements of helices, loops and other structural elements. It is difficult to study these dynamic conformational transitions using conventional methods of structure analysis. Time-resolved fluorescence resonance energy transfer (tr-FRET) is a powerful technique for the study of RNA conformational transitions because it can provide structural, thermodynamic and kinetic information. In this proposal, tr-FRET and related spectroscopic methods will be used to study biologically-relevant RNA conformational transitions in three different systems.
The specific aims are: 1. Analyze tertiary structure formation in the hairpin ribozyme. Elucidate the structural and thermodynamic basis for efficient docking of the substrate-binding and catalytic domains of the ribozyme-substrate complex and characterize changes in base pairing and stacking that occur during domain docking. 2. Elucidate the energetic basis for ion- and protein-induced folding of RNA three-way junctions. Determine the global conformations of three-way junctions from 16 S and 5 S rRNAs and establish how the binding of metal ions and proteins are linked to conformational changes within the RNA. 3. Determine the global structure of a large RNA fragment from the Rev Response Element. Establish whether binding of HIV-1 Rev and Rev-Rev multimerization on the RNA induce a rearrangement of helices. The results of this research will reveal how the structure of a helical junction can direct the docking of distant RNA domains and stabilize a biologically active tertiary structure. The findings will also contribute to an understanding of how proteins and metal ion cofactors can regulate the biological activity of RNA. In addition, the results will aid in the design of improved therapeutic ribozymes and contribute to the development of drugs directed against RNA targets.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
3R01GM058873-04S1
Application #
6891987
Study Section
Molecular and Cellular Biophysics Study Section (BBCA)
Program Officer
Lewis, Catherine D
Project Start
2000-04-01
Project End
2005-04-30
Budget Start
2003-04-01
Budget End
2005-04-30
Support Year
4
Fiscal Year
2004
Total Cost
$78,209
Indirect Cost
Name
Scripps Research Institute
Department
Type
DUNS #
781613492
City
La Jolla
State
CA
Country
United States
Zip Code
92037
Pljevaljcic, Goran; Robertson-Anderson, Rae; van der Schans, Edwin et al. (2012) Analysis of RNA folding and ribonucleoprotein assembly by single-molecule fluorescence spectroscopy. Methods Mol Biol 875:271-95
Pljevaljcic, Goran; Millar, David P (2008) Single-molecule fluorescence methods for the analysis of RNA folding and ribonucleoprotein assembly. Methods Enzymol 450:233-52
Pljevaljcic, Goran; Klostermeier, Dagmar; Millar, David P (2005) The tertiary structure of the hairpin ribozyme is formed through a slow conformational search. Biochemistry 44:4870-6
Pljevaljcic, Goran; Millar, David P; Deniz, Ashok A (2004) Freely diffusing single hairpin ribozymes provide insights into the role of secondary structure and partially folded states in RNA folding. Biophys J 87:457-67
Klostermeier, Dagmar; Millar, David P (2002) Energetics of hydrogen bond networks in RNA: hydrogen bonds surrounding G+1 and U42 are the major determinants for the tertiary structure stability of the hairpin ribozyme. Biochemistry 41:14095-102
Ryder, S P; Oyelere, A K; Padilla, J L et al. (2001) Investigation of adenosine base ionization in the hairpin ribozyme by nucleotide analog interference mapping. RNA 7:1454-63
Klostermeier, D; Millar, D P (2001) RNA conformation and folding studied with fluorescence resonance energy transfer. Methods 23:240-54
Klostermeier, D; Millar, D P (2001) Time-resolved fluorescence resonance energy transfer: a versatile tool for the analysis of nucleic acids. Biopolymers 61:159-79
Klostermeier, D; Millar, D P (2001) Tertiary structure stability of the hairpin ribozyme in its natural and minimal forms: different energetic contributions from a ribose zipper motif. Biochemistry 40:11211-8
Klostermeier, D; Millar, D P (2000) Helical junctions as determinants for RNA folding: origin of tertiary structure stability of the hairpin ribozyme. Biochemistry 39:12970-8