Locating, characterizing, and determining the effect of metal ions in the hammerhead ribozyme /
The hammerhead ribozyme is a catalytic RNA that performs a site-specific bond cleavage and is active in the presence of divalent cations including Mg²⁺, Mn²⁺, Cd²⁺, and Co²⁺. The relative affinities of these metal ions follows Mn²⁺~ Co²⁺ >Cd²⁺ >>Mg²⁺. Maximum rates of cleavage result from...
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| Format: | Thesis Book |
| Language: | English |
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[Place of publication not identified] :
[publisher not identified] ;
2001.
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| Subjects: | |
| Online Access: | http://proxy.library.tamu.edu/login?url=http://proquest.umi.com/pqdweb?did=726102871&sid=1&Fmt=2&clientId=2945&RQT=309&VName=PQD |
| Summary: | The hammerhead ribozyme is a catalytic RNA that performs a site-specific bond cleavage and is active in the presence of divalent cations including Mg²⁺, Mn²⁺, Cd²⁺, and Co²⁺. The relative affinities of these metal ions follows Mn²⁺~ Co²⁺ >Cd²⁺ >>Mg²⁺. Maximum rates of cleavage result from several factors including high affinity, low pK[a], and lower [][Hyd], the best combination of which is found in Mn²⁺. Previous reports show that 8-9 Mn²⁺ bind to the hammerhead in 2 types of sites (Horton et al., 1998) with ~3-4 high affinity sites (K[d] ~ 4[]M) and 4-5 lower affinity sites (K[d] ~ 400 []M). Mutations to the conserved core of the hammerhead result in loss of high affinity sites localizing the high affinity sites to the conserved core. The metal ions in the high affinity sites can affect the apparent affinity of a metal ion involved in cleavage. The highest affinity Mn²⁺ site in the hammerhead has been characterized using EPR, ESEEM, EXAFS and ³¹P NMR when there is a phosphorothioate at A9. The highest affinity Mn²⁺ coordinates to nitrogen and to a phosphate. When Mn²⁺ binds A9-S[Rp], there is a change in the phosphate coordination and the activity is diminished compared to wild type or the A9-S[Sp]. Therefore, in A9-S[Rp], the altered coordination may cause a conformational change, resulting in a "slowly cleaving" ribozyme. Spectroscopic studies show that Cd²⁺ displaces the Mn²⁺ at the phosphorothioate- substituted A9 site. Addition of Cd²⁺ to A9-S[Rp] returns the cleavage rate to near that of wild type and could indicate that Cd²⁺ binding relieves any deleterious conformational change. A phosphorothioate substitution is a common experiment to locate potential metal binding sites in RNA, however, there have been few studies that investigate the structural and electronic differences between phosphates, phosphorothioates, and thiolates. Theoretical models reveal some fundamental differences between phosphates, phosphorothioates, and thiolates and their interactions with Cd²⁺. In general, a thiolate-Cd interaction is highly covalent, a phosphorothioate-Cd interaction is somewhat covalent, and a phosphate-Cd interaction is purely electrostatic. These differences help explain some of the unique spectroscopic characteristics of phosphorothioates. |
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| Item Description: | Vita. "Major Subject: Chemistry". |
| Physical Description: | xvi, 242 leaves : illustrations ; 28 cm. Issued also on microfiche from University Microfilm Inc. |
| Bibliography: | Includes bibliographical references (leaves 222-234). |