Investigation of metal binding sites in the hammerhead ribozyme by paramagnetic resonance techniques /
The hammerhead ribozyme, small, well-characterized RNA molecule, undergoes a phosphodiester bond self-cleavage reaction in the presence of divalent metal ions. Information about the structural roles of the divalent metals can provide an understanding of how they support activity in the ribozyme. Mn²...
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| Format: | Thesis Book |
| Language: | English |
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[Place of publication not identified] :
[publisher not identified] ;
1999.
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| Subjects: | |
| Online Access: | http://proxy.library.tamu.edu/login?url=http://proquest.umi.com/pqdweb?did=731681851&sid=1&Fmt=2&clientId=2945&RQT=309&VName=PQD |
| Summary: | The hammerhead ribozyme, small, well-characterized RNA molecule, undergoes a phosphodiester bond self-cleavage reaction in the presence of divalent metal ions. Information about the structural roles of the divalent metals can provide an understanding of how they support activity in the ribozyme. Mn²⁺ has been employed as a paramagnetic probe, making possible the application of paramagnetic techniques such as electron paramagnetic resonance (EPR), electron spin echo envelope modulation (ESEEM), and electron nuclear double resonance (ENDOR) to the study of Mn²⁺ binding in the hammerhead ribozyme. Low temperature X-band EPR studies have provided a spectroscopic indicator for Mn²⁺ bound by the ribozyme. Low temperature relaxation studies demonstrated that the four high affinity sites existing within the ribozyme under conditions of 100 mM NaCl are each ~6 [] to ~15 [] apart. These studies did not provide evidence to support a dinuclear Mn²⁺ site as proposed in some mechanisms. A cw Q-band ENDOR spectrometer was built locally to investigate metal binding in biomolecules. ENDOR, a technique used to provide information about the coordination environment of the paramagnetic center, was employed to investigate a Mn²⁺-ribozyme sample under conditions of 1 M NaCl where a single Mn²⁺ site is populated. The ENDOR studies provided evidence for an inner sphere phosphate group, nonexchangeable protons, and water molecules in the Mn²⁺ coordination environment. One nonexchangeable proton was proposed to be a purine C8-proton, indicating a Mn²⁺ inner sphere coordination to a purine N7. Inner verses outer sphere phosphate coordination assignments were confirmed using Mn-ATP and Mn-GMP model systems. ESEEM, a pulsed EPR technique, provides information about the metal coordination environment that is similar to ENDOR but more sensitive to weak hyperfine couplings. The three-pulse X-band ESEEM data of Mn²⁺-ribozyme and Mn²⁺-nucleotide model complexes containing both naturally abundant ¹⁴N and labeled ¹⁵N nuclei verified the existence of a Mn²⁺-N7 coordination to a guanine base in the ribozyme. Correlating the characteristics of the Mn²⁺ bindings site determined by the ENDOR and ESEEM studies with the metal binding sites determined by crystallographic studies implicated the A9/G10.1 site observed in the crystallographic studies as being populated in these studies. |
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| Item Description: | Vita. "Major Subject: Chemistry". |
| Physical Description: | xii, 128 leaves : illustrations ; 28 cm. Issued also on microfiche from University Microfilm Inc. |
| Bibliography: | Includes bibliographical references (leaves 123-127). |