Investigation of metal binding properties in the hammerhead ribozyme and tetraloop hairpins by various spectroscopic techniques /
The hammerhead ribozyme is a well-characterized RNA molecule capable of catalyzing a site-specific phosphodiester bond cleavage reaction in the presence of divalent cations. The metal-RNA interactions that promote this activity are not well understood. Metal-RNA interactions are investigated in thi...
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| Format: | Thesis Book |
| Language: | English |
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[Place of publication not identified] :
[publisher not identified] ;
2000.
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| Subjects: | |
| Online Access: | http://proxy.library.tamu.edu/login?url=http://proquest.umi.com/pqdweb?did=731990111&sid=1&Fmt=2&clientId=2945&RQT=309&VName=PQD |
| Summary: | The hammerhead ribozyme is a well-characterized RNA molecule capable of catalyzing a site-specific phosphodiester bond cleavage reaction in the presence of divalent cations. The metal-RNA interactions that promote this activity are not well understood. Metal-RNA interactions are investigated in this work using a variety of methods, including electron paramagnetic resonance (EPR) spectroscopy, circular dichroism spectroscopy and optically and calorimetrically detected thermal denaturation studies. Electron paramagnetic resonance spectroscopy of Mn²⁺is used to determine the numbers and affinities of Mn²⁺ bound to several hammerhead ribozyme constructs. In 0.1 M NaCl the hammerhead ribozyme binds Mn²⁺ at two 'types' of sites with affinities of K[] < 10 []M for the 'tight' type 1 sites and K[] > 350 []M for the 'weak' type 2 sites. Metal binding was found to be highly dependent on NaCl concentration. Activity studies show maximal activity is reached upon saturation of these sites. Cobalt hexammine can be used as a ligand-exchange inert probe for magnesium hexahydrate. Low concentrations of cobalt hexammine promote the release from the hammerhead of all but one bound Mn²⁺. This release coincides with a 15-fold reduction in rate, but not complete inhibition. This suggests that only a limited number of inner-sphere metal-RNA contacts are required for hammerhead activation. The ligand environment of a single bound Mn²⁺ ion in 1 M NaCl was determined using EPR/ENDOR and ESEEM studies with a ¹⁵N guanine-labeled hammerhead. These studies showed that the Mn²⁺ ion is bound directly to a phosphate oxygen and N7 of a guanosine base in the hammerhead ribozyme consistent with the A9/G10.1 site predicted by X-ray crystallography. Thermal denaturation studies of various hammerhead constructs provide enthalpic contributions for core formation in the WT hammerhead ribozyme. A tentative unfolding pathway can be assigned from these data. Thermal denaturation studies of a 12-nucleotide ⁵G̀AAA³ ́hairpin shows a significant stabilization upon addition of a single site-specific phosphorothioate substitution. Sulfur substitution of an oxygen at the R[]-A7 position in the tetraloop results in a net 2.9 kcal mol⁻¹ stabilization of this hairpin. |
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| Item Description: | Vita. "Major Subject: Chemistry". |
| Physical Description: | xiii, 183 leaves : illustrations ; 28 cm. Issued also on microfiche from University Microfilm Inc. |
| Bibliography: | Includes bibliographical references (leaves 173-181). |