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Han, Joonhee

Microbiology and Molecular Genetics

2005

PhD

The hammerhead (HH) ribozyme is a small catalytic RNA, which was derived from the positive strand of the tobacco ringspot virus satellite RNA. The hammerhead ribozyme catalyzes a sequence-specific phosphodiester bond cleavage reaction, generating 2', 3'-cyclic phosphate and 5'-hydroxyl group termini. The three-dimensional structure of the hammerhead ribozyme was revealed by X-ray crystallography. It has, however, been found that the crystal structures are not consistent with the biochemical data, suggesting that the crystal structure does not seem to represent the transition state conformation in which in-line attack can occur. It is obvious that, therefore, substantial structural changes at the active site are required for catalysis. It has also been found that the role of divalent metal ions is to stabilize the conformation of the ribozyme-substrate complex for catalysis, rather than act as catalysts during the chemical cleavage reaction.
With those backgrounds, we hypothesized that certain nucleotides at the active site of the hammerhead ribozyme could be directly involved in the chemical cleavage reaction and conformational changes leading to the transition state. In this thesis, using base substitution, hydroxyl radical footprinting and crosslinking assays, I present evidence that these catalytic groups are the N1s of two conserved nucleobases within the catalytic core, G8 and G12 and that the conformational change into the transition state requires a close approach of helices 1 and 2, consequently resulting in a closely packed structure or, at least, that the interaction between helices 1 and 2 might play an important structural role in the folding of the hammerhead ribozyme. The roles of the essential nucleotides (G8 and G12) for catalysis and conformational changes are also elucidated and magnesiuminduced folding process are studied using the hammerhead ribozymes.