A conserved bulged adenosine in a peripheral duplex of the antigenomic HDV self-cleaving RNA reduceskinetic trapping of inactive conformations.
In the ribozyme of hepatitis delta virus antigenomic RNA, two short duplexes, P2 and P2a, stabilize the active self-cleaving structure. However, P2a also promotes kinetic trapping of non-native structures. A bulged adenosine (A14) separates P2a and P2; this bulged A is conserved in clinical isolates of HDV but is unlikely to be physically close to the cleavage site phosphate in the ribozyme structure. Removing the bulge did not significantly slow the rate of cleavage but slowed the conversion of inactive to active conformations. In the absence of the bulged A, inactive conformations required higher urea concentrations or higher temperatures to be activated. Thus, the bulged-nucleotide in the P2-P2a duplex did not provide an essential kink or hinge between P2 and P2a that was required for cleavage activity but, rather, increased the rate of refolding from an inactive to an active ribozyme structure. These data also suggest a model in which P2 and P2a form a coaxial stacked helix of 9 bp, the most likely arrangement being one in which P2-P2a is roughly parallel to P1.
Perrotta
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Department of Biochemistry, Duke University Medical School, Durham, NC 27710, USA.
Nikiforova
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Been
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D000074183:ATPases Associated with Diverse Cellular Activities; D000241:Adenosine / Q000737:chemistry*; D001483:Base Sequence; D017124:Conserved Sequence; D006023:Glycoproteins / Q000378:metabolism; D003698:Hepatitis Delta Virus / Q000235:genetics*; D007700:Kinetics; D008565:Membrane Proteins; D008969:Molecular Sequence Data; D009154:Mutation; D009690:Nucleic Acid Conformation; D017510:Protein Folding; D011498:Protein Precursors / Q000378:metabolism; D016337:RNA, Catalytic / Q000737:chemistry / Q000235:genetics*; D012367:RNA, Viral / Q000737:chemistry*; D012965:Sodium Chloride / Q000378:metabolism; D013095:Spermidine / Q000378:metabolism; D013329:Structure-Activity Relationship
D006023:Glycoproteins; D008565:Membrane Proteins; D011498:Protein Precursors; D016337:RNA, Catalytic; D012367:RNA, Viral; D012965:Sodium Chloride; D000074183:ATPases Associated with Diverse Cellular Activities; C111808:PEX1 protein, human; D000241:Adenosine; D013095:Spermidine