Point and compensation mutations to evaluate essential stem structures of genomic HDV ribozyme

In elucidating the part played by the essential stem structures (I, II, and III) in the self-cleavage activity of genomic HDV ribozyme, several point and compensation variants were constructed on pseudoknot-like structure by site-directed mutagenesis. The self-cleavage activities of these variants indicated that stems I and III were essential for the activity by forming Watson-Crick base pairs. On the other hand, disruption of A704:U767 had little influence on the cleavage activity, indicating that it is not essential in forming an active structure. Also, our V1 nuclease probing studies showed that the A704U and HDV88 variants have a sensitivity similar to the nuclease, and major cuts are visible in the stem I and stem II regions. Thus, stem I and stem II regions are maintained together with stem III regions in both molecules. These results and our earlier site-directed mutagenesis studies strongly support a pseudoknot-like structure for the genomic HDV ribozyme.