Hepatitis delta virus ribozymes fold to generate a solvent-inaccessible core with essential nucleotides near the cleavage site phosphate.
Self-cleaving sequences or ribozymes from the hepatitis delta virus (HDV) genomic RNA and its complement form similar secondary structures that suggest a core region and potential active site composed of "single-stranded" sequences. However, there is little data on tertiary interactions in these ribozymes, therefore structural features were investigated using cross-linking and hydroxyl radical cleavage. Cross-links in cis and trans forms of the antigenomic RNA were generated using the photoactivatable azidophenacyl group tethered to the cleavage site phosphate. Specific cross-links formed to J4/2, and to the 3' sides of P3 and L3. Different sites were cross-linked in low salt or monovalent cations versus divalent cations, suggesting a metal ion-dependent conformational change near the cleavage site. The solvent-inaccessible regions of both the genomic and antigenomic ribozymes were revealed by cleavage in Fe(II)-EDTA. In Mg2+, backbone segments most strongly protected from solvent-based hydroxyl radicals were mapped to J4/2 and parts of L3. Similar patterns of protection were seen in trans-acting ribozymes bound to a product oligonucleotide. These data provide evidence for a common tertiary structure for the HDV ribozymes. They would be consistent with a model in which the end of P1, including the cleavage site phosphate and the nucleotide 5' to the cleavage site, is positioned in an active site pocket or cleft formed by the three single-stranded regions, L3, J4/2, and J1/4.
Rosenstein
S P
SP
Department of Biochemistry, Duke University Medical Center, Durham, North Carolina 27710, USA.
Been
M D
MD
D001386:Azides / Q000378:metabolism; D001483:Base Sequence; D001665:Binding Sites; D003432:Cross-Linking Reagents; D004492:Edetic Acid / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D005296:Ferrous Compounds / Q000494:pharmacology; D003698:Hepatitis Delta Virus / Q000235:genetics*; D017665:Hydroxyl Radical / Q000378:metabolism; D008274:Magnesium / Q000494:pharmacology; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D010782:Photolysis; D016337:RNA, Catalytic / Q000737:chemistry* / Q000378:metabolism; D012367:RNA, Viral / Q000737:chemistry* / Q000378:metabolism; D014466:Ultraviolet Rays
D001386:Azides; D003432:Cross-Linking Reagents; D005296:Ferrous Compounds; D016337:RNA, Catalytic; D012367:RNA, Viral; C055526:Fe(II)-EDTA; D017665:Hydroxyl Radical; D004492:Edetic Acid; D008274:Magnesium