The three-dimensional structure of RNA is critical to its cellular function. Catalytic RNA molecules, called ribozymes, fold into complex globular structures to produce active sites that promote chemical reactions independent of protein facilitation. Although the molecular basis for such structures is currently unknown, their formation involves the assembly of short helical elements via direct RNA-RNA and metal-mediated contacts. An understanding of the molecular interactions that give to correctly folded RNAs is fundamental to understanding the structural and mechanistic principles of RNA catalysis. A detailed knowledge of the principles of higher order folding in RNA will enable rational design of efficient ribozymes for therapeutic purposes and will illuminate the role of RNA in such fundamental biological processes as protein biosynthesis and messenger RNA splicing. Like proteins, large catalytically active RNA molecules are often composed of independently folding structural domains. In the well- studied group I self-splicing introns, the catalytic core is comprised of helical components residing in two separate domains. The crystal structure of one of these, the independently folding P4-P6 domain, revealed that conserved helices of the core lie parallel to helices in an extended subdomain. In addition to two specific sets of tertiary contacts, divalent metal ions and 2' hydroxyl groups of riboses in the RNA backbone stabilize this remarkably close helical packing. This exciting structure also provides tantalizing clues to the organization of the complete intron catalytic core, and is thus the basis for probing structural interactions both within the P4-P6 domain and between P4-P6 and the rest of the intron.
The specific aims of this proposal are threefold: 1. Determine the affinity and specificity of magnesium ion binding sites clustered in an adenosine-rich corkscrew motif, the A-rich bulge, that is central to P4-P6 domain folding. 2. Define the classes of divalent metal ion binding sites in the P4-P6 crystal structure, and correlate them with functional data for P4-P6 folding and intron catalysis. 3. Measure the energetic contributions of individual tertiary contacts within the P4- P6 domain, involving magnesium ions, ribose 2' hydroxyl groups and nucleotide bases, to domain structure and global intron folding and catalysis.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM056361-02
Application #
2750168
Study Section
Molecular and Cellular Biophysics Study Section (BBCA)
Project Start
1997-08-01
Project End
2002-07-31
Budget Start
1998-08-01
Budget End
1999-07-31
Support Year
2
Fiscal Year
1998
Total Cost
Indirect Cost
Name
Yale University
Department
Biochemistry
Type
Schools of Medicine
DUNS #
082359691
City
New Haven
State
CT
Country
United States
Zip Code
06520
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Doherty, E A; Herschlag, D; Doudna, J A (1999) Assembly of an exceptionally stable RNA tertiary interface in a group I ribozyme. Biochemistry 38:2982-90