Experimental evidence for the secondary structure of the hepatitis delta virus ribozyme

Specific features of a model for the secondary structure of the self-cleaving RNA sequences (ribozymes) of hepatitis delta virus were rigorously tested. Using a self-cleaving form of the antigenomic sequence, mutations were made in the 5' and 3' sequences of each of four duplex regions within the proposed ribozyme structure. Precursor RNA from each variant sequence was prepared and the kinetics of cleavage in 10 mM Mg2+ at 37 degrees was examined. The data was quantified to determine an end point and a first-order rate constant for cleavage with each mutant by fitting the data to the exponential form of the first-order rate equation. With regard to the final extent of cleavage, most mutations in these regions appeared to have little effect, however, the kinetics indicated that disruption of the potential for basepairing resulted in dramatic decreases in the rate constant for cleavage. These results are consistent with the idea that most of the mutations affected ribozyme activity rather than an equilibrium between precursor and cleavage products. Mutations that reduced rates were compensated by changes that restored the potential for Watson-Crick pairing. Ribonuclease probing of ribozyme variants containing mismatches and compensatory changes allowed direct correlation of structural changes with the mutations. This provided an independent validation of the functional kinetic assay. Thus, site-directed mutagenesis was consistent with a proposed ribozyme secondary structure containing 4 distinct base-paired regions.