Conformational changes and metal -ion binding in the hepatitis delta virus ribozyme

2004 2004

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Abstract (summary)

The hepatitis delta virus (HDV) ribozyme is among the class of small ribozymes that catalyze a self-cleavage reaction critical to the replication of a small RNA genome. In the present work, several fluorescence techniques were tested to investigate the catalytic strategies applied by the HDV ribozyme. Fluorescence resonance energy transfer (FRET) and 2-aminopurine (AP) fluorescence were employed to examine the role that global and local conformational changes on the reaction pathway of the HDV ribozyme have in catalysis. Using fluorescence resonance energy transfer on a synthetic trans-cleaving form of the ribozyme, we were able to directly observe substrate binding and dissociation. Steady-state and time-resolved FRET experiments in solution and in non-denaturing gels revealed that the substrate (precursor) complex is slightly more compact than the free ribozyme, yet becomes significantly extended upon cleavage and product complex formation. By modifying the solvent exposed nucleotide G76 of the trefoil turn of the synthetic trans-cleaving HDV ribozyme to the fluorescent 2-aminopurine (AP), we directly monitored local conformational changes in the catalytic core that accompany catalysis. Additionally, the lanthanide metal ion terbium(III) was used to footprint the precursor and product solution structures of the cis-acting antigenomic HDV ribozyme. Subtle, yet significant differences between the terbium(III) footprinting patterns of the precursor and product forms of the antigenomic HDV ribozyme are consistent with differences in conformation. In addition, UV melting profiles provided evidence for a less tight tertiary structure in the precursor. In both the precursor and product, high-affinity terbium(III) binding sites were observed in joining sequence J4/2 and loop L3, which are key structural components forming the catalytic core of the HDV ribozyme, as well as in several single-stranded regions such as J1/2 and the L4 tetraloop. Sensitized luminescence spectroscopy confirmed that there are at least two affinity classes of Tb3+ binding sites. These experiments show that the precursor and product forms of the HDV ribozyme are structurally distinct and have different metal ion affinities. The presented biochemical and biophysical studies provide important new insights into the relationship between structure and function of the HDV ribozyme.

Indexing (details)

0487: Biochemistry
Identifier / keyword
Pure sciences; Hepatitis delta virus; Metal-ion binding; Ribozyme
Conformational changes and metal -ion binding in the hepatitis delta virus ribozyme
Harris, Dinari A.
Number of pages
Publication year
Degree date
School code
DAI-B 65/10, Dissertation Abstracts International
Place of publication
Ann Arbor
Country of publication
United States
049609789X, 9780496097890
Walter, Nils G.
University of Michigan
University location
United States -- Michigan
Source type
Dissertations & Theses
Document type
Dissertation/thesis number
ProQuest document ID
Database copyright ProQuest LLC; ProQuest does not claim copyright in the individual underlying works.
Document URL
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