A toggle duplex in hepatitis delta virus self-cleaving RNA that stabilizes an inactive and a salt-dependent pro-active ribozyme conformation.
The antigenomic RNA of hepatitis delta virus (HDV) can form a short duplex, P2a, in which a four-nucleotide sequence within the self-cleaving domain pairs with a sequence just outside the previously defined 3'-boundary of the ribozyme. Both sequences that would participate in forming P2a were previously determined to be non-essential for self-cleavage activity. Ribozymes able to form P2a were less active than those lacking the 3' P2a sequence when preincubated under the standard low-Na+ conditions. Chemical probing of the RNA correlated base-pairing in P2a with this inhibition. Furthermore, mutagenesis and 3' truncation experiments mapped the inhibitory sequence to P2a. However, raising the NaCl concentration in the preincubation prior to adding Mg2+ reversed the inhibitory effect. Moreover, with NaCl preincubation, the P2a-containing ribozyme was more active than an otherwise identical ribozyme lacking the 3' P2a sequence. Non-denaturing gels provided evidence for alternative conformations of the P2a-containing precursor with only the faster-migrating species correlating with the active form. A difference in the temperature-dependence for the rate of cleavage of the P2a-containing ribozyme with and without NaCl, together with a difference in the melting behavior of the RNA in NaCl with and without P2a, suggested that P2a favors the native structure in NaCl. Many derivatives of the HDV ribozymes form inactive conformers; however, this study reveals details of a specific structure that stabilizes both inactive and active conformations of the HDV ribozyme.
Perrotta
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Department of Biochemistry, Duke University Medical Center, Durham, NC 27710, USA.
Been
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MD
D001483:Base Sequence; D001665:Binding Sites; D004795:Enzyme Stability; D003698:Hepatitis Delta Virus / Q000201:enzymology* / Q000235:genetics; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D016337:RNA, Catalytic / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D012367:RNA, Viral / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D012965:Sodium Chloride; D014508:Urea
D016337:RNA, Catalytic; D012367:RNA, Viral; D012965:Sodium Chloride; D014508:Urea