A role for upstream RNA structure in facilitating the catalytic fold of the genomic hepatitis delta virus ribozyme

Durga M. Chadalavada, Scott M. Knudsen, Shu Ichi Nakano, Philip C. Bevilacqua

Research output: Contribution to journalArticle

68 Scopus citations

Abstract

Hepatitis delta virus (HDV) has a circular RNA genome that replicates by a double rolling-circle mechanism. The genomic and antigenomic versions of HDV contain a ribozyme that undergoes cis-cleavage, thereby processing the transcript into unit-length monomers. A genomic HDV transcript containing 30 nucleotides immediately upstream of the cleavage site was found to have attenuated self-cleavage. Structure mapping and site-directed mutagenesis revealed an inhibitory stretch consisting of upstream nucleotides -24 to -15 that forms a long-range pairing, termed Alt 1, with the 3' strand of P2 (P2(3')) located at the very 3'-end of the ribozyme. Two other alternative pairings were found, Alt 2, which involves upstream nucleotide-ribozyme interactions, and Alt 3, which involves ribozyme-ribozyme interactions. Self-cleavage was rescued 2700 to 20,000-fold by adding DNA oligomers, which sequester the -24/-15 inhibitory stretch in trans. Surprisingly, co-transcriptional self-cleavage occurs when the number of upstream nucleotides is increased to 54. Computer prediction and structure mapping support the existence of an unusually stable upstream hairpin involving nucleotides -54 to -18, termed P(-1)/L(-1), which sequesters the majority of the -24/-15 inhibitory stretch in cis. This hairpin is followed by a stretch of single-stranded pyrimidine-rich nucleotides, termed J(-1/1). Sequence comparison suggests that the P(-1)/L(-1)/J(-1/1) motif is conserved among known genomic HDV isolates, and that the J(-1/1) stretch is conserved among antigenomic HDV isolates. Lastly, the secondary structure of the Alt 1-containing ribozyme provides insight into possible folding intermediates of the ribozyme. (C) 2000 Academic Press.

Original languageEnglish (US)
Pages (from-to)349-367
Number of pages19
JournalJournal of Molecular Biology
Volume301
Issue number2
DOIs
StatePublished - Aug 11 2000

All Science Journal Classification (ASJC) codes

  • Structural Biology
  • Molecular Biology

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