Metal-phosphate interactions in RNA investigated by ³¹P NMR spectroscopy and phosphorothioate modifications /
Metal ions play an important role in both structure and function in many RNA molecules. ³¹P NMR spectroscopy, in conjunction with phosphorothioate modifications, can be used to probe for specific metal-phosphate interactions. Effects of metal binding to small models containing phosphates and thiop...
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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=727853111&sid=1&Fmt=2&clientId=2945&RQT=309&VName=PQD |
| Summary: | Metal ions play an important role in both structure and function in many RNA molecules. ³¹P NMR spectroscopy, in conjunction with phosphorothioate modifications, can be used to probe for specific metal-phosphate interactions. Effects of metal binding to small models containing phosphates and thiophosphates indicated that a M²⁺-phosphate interaction results in a downfield shift of the ³¹P NMR resonance and a M²⁺-thiophosphate interaction results in an upfield shift. Theoretical calculations were performed in order to try to understand the reasons for this directional shift with phosphate vs. thiophosphate. A ³¹P NMR investigation of a 12-nucleotide hairpin with a loop sequence of 5'-GAAA-3' provided evidence for a metal-phosphate interaction with the metal directly coordinating to the phosphate 5' to the second A in the loop (A7). In addition, these studies indicated that there is perturbation in the structure with a specific Rp-phosphorothioate modification 5' to A7 (A7-Rp), compared to WT. The structural differences in WT and A7-Rp hairpin are currently being pursued using two-dimensional NMR techniques. Initial data are consistent with a model in which there is a structural rearrangement in the loop and stem regions of the A7-Rp hairpin. The hammerhead ribozyme is an RNA molecule capable of a site specific phosphodiester bond cleavage reaction. The rate of this bond cleavage is greatly enhanced in the presence of metal ion(s). One proposed role for metal ions in the cleavage reaction involves coordination to the pro-R oxygen of the scissile phosphate. An Rp- or Sp- phosphorothioate modification was placed at the scissile phosphate and ³¹P NMR studies were used to investigate the effects with added Mg²⁺/Cd²⁺. Spectroscopic evidence for a direct metal-phosphate interaction with the pro-R oxygen at the scissile phosphate of the hammerhead ribozyme is provided by ³¹P NMR data, supporting this proposed role for a metal ion in the mechanism of cleavage. The utility of ³¹P NMR and phosphorothioate modifications as a tool to investigate metal-phosphate interactions in nucleic acids is demonstrated. The results of this work show that ³¹P NMR spectroscopy can be used to provide evidence for structural or functional roles of metal ions in RNA molecules. |
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| Item Description: | Vita. "Major Subject: Chemistry". |
| Physical Description: | xii, 193 leaves : illustrations ; 28 cm. Issued also on microfiche from University Microfilm Inc. |
| Bibliography: | Includes bibliographical references (leaves 181-191). |