The hammerhead ribozyme (HHRz) catalyzes a site-specific
phosphodiester bond cleavage reaction that is enhanced by the presence of
metal cations. Metal cations are thought to aid in the folding and possibly the
catalytic mechanism of this ribozyme. The goal of this research is to characterize
the activities and metal interactions of minimal and extended HHRz constructs
using kinetic and spectroscopic studies.
Metal binding to the cleavage site of the HHRz was probed using 31P
NMR to monitor Cd2+ titrations of HHRzs with a phosphorothioate modification at
the cleavage site. Either a 2'-F or a 2'-NH2 substitution at the nucleophile
position was used to block cleavage. With a 2'-F, no metal binding to the
cleavage site phosphate was observed. However, with a 2'-NH2 substitution, a
large change in 31P chemical shift of the phosphorothioate peak suggests Cd2+
binding. A 2'-NH2 is a potential metal ligand, but a 2'-F is not. This suggests that
a metal ion binds to the cleavage site phosphate when the 2' nucleophile
position also provides a ligand. Minimal HHRzs with only one stem loop structure show little activity in
presence of physiologically relevant concentrations of divalent cations. A kinetic
and thermodynamic characterization of an extended HHRz derived from
Schistosoma mansoni with loops in stems I and II was performed. High catalytic
activity was observed with low concentrations of divalent cations, and loss of
potential loop-loop interactions resulted in a large decrease in activity.
An electrostatic surface plot of a HHRz crystal structure revealed an area
of high negative electrostatic potential in the cleft between stems I and II with
contributions from nucleotides U7, A6, and C17 of the HHRz that could serve to
trap metal ions. To probe this putative metal site, kinetic studies of HHRz
constructs with phosphorothioate substitutions 5' to U7 or C17 or with an A6 2'-
OMe substitution were performed. Results of these studies suggest that a metal
interaction at this site would include direct coordination to A6 2'-OH, but indirect
interactions with the phosphates.
| Identifer | oai:union.ndltd.org:tamu.edu/oai:repository.tamu.edu:1969.1/4925 |
| Date | 25 April 2007 |
| Creators | Osborne, Edith Marie |
| Contributors | DeRose, Victoria J., Scholtz, J. Martin |
| Publisher | Texas A&M University |
| Source Sets | Texas A and M University |
| Language | en_US |
| Detected Language | English |
| Type | Book, Thesis, Electronic Dissertation, text |
| Format | 5709241 bytes, electronic, application/pdf, born digital |
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