A conserved bulged adenosine in a peripheral duplex of the antigenomic HDVself-cleaving RNA reduces kinetic trapping of inactive conformations

In the ribozyme of hepatitis delta virus antigenomic RNA, two short duplexe s, P2 and P2a, stabilize the active self-cleaving structure. However, P2a a lso 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 phospha te in the ribozyme structure. Removing the bulge did not significantly slow the rate of cleavage but slowed the conversion of inactive to active confo rmations, In the absence of the bulged A, inactive conformations required h igher urea concentrations or higher temperatures to be activated. Thus, the bulged-nucleotide in the P2-P2a duplex did not provide an essential kink o r hinge between P2 and P2a that was required for cleavage activity but, rat her, 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 coax ial stacked helix of 9 bp, the most likely arrangement being one in which P 2-P2a is roughly parallel to P1.