title: Local conformational changes in the catalytic core of the trans-acting hepatitis delta virus ribozyme accompany catalysis.
abstract: The hepatitis delta virus (HDV) is a human pathogen and satellite RNA of the hepatitis B virus. It utilizes a self-cleaving catalytic RNA motif to process multimeric intermediates in the double-rolling circle replication of its genome. Previous kinetic analyses have suggested that a particular cytosine residue (C(75)) with a pK(a) close to neutrality acts as a general acid or base in cleavage chemistry. The crystal structure of the product form of a cis-acting HDV ribozyme shows this residue positioned close to the 5'-OH leaving group of the reaction by a trefoil turn in the RNA backbone. By modifying G(76) of the trefoil turn of a synthetic trans-cleaving HDV ribozyme to the fluorescent 2-aminopurine (AP), we can directly monitor local conformational changes in the catalytic core. In the ribozyme-substrate complex (precursor), AP fluorescence is strongly quenched, suggesting that AP(76) is stacked with other bases and that the trefoil turn is not formed. In contrast, formation of the product complex upon substrate cleavage or direct product binding results in a significant increase in fluorescence, consistent with AP(76) becoming unstacked and solvent-exposed as evidenced in the trefoil turn. Using AP fluorescence and fluorescence resonance energy transfer (FRET) in concert, we demonstrate that this local conformational change in the trefoil turn is kinetically coincidental with a previously observed global structural change of the ribozyme. Our data show that, at least in the trans-acting HDV ribozyme, C(75) becomes positioned for reaction chemistry only along the trajectory from precursor to product.
mesh_terms: D015075:2-Aminopurine / Q000378:metabolism; D001665:Binding Sites; D020134:Catalytic Domain; D003698:Hepatitis Delta Virus / Q000201:enzymology / Q000235:genetics / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D009690:Nucleic Acid Conformation; D016337:RNA, Catalytic / Q000378:metabolism*; D012367:RNA, Viral / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D015533:Transcriptional Activation
chemical_list: D016337:RNA, Catalytic; D012367:RNA, Viral; D015075:2-Aminopurine