Sequential folding of the genomic ribozyme of the hepatitis delta virus: Structural analysis of RNA transcription intermediates

The structures of the model oligoribonucleotides that mimic the consecutive stages in the transcription of genomic HDV ribozyme have been analyzed by the Pb2+-induced cleavage method, partial digestion with specific nucleases and chemical probing. In the transcription intermediates, the P1 and P4 he lical segments are found to be present in the final folded forms in which t hey exist in the full-length transcript. However, the region corresponding to the central hairpin forms another thermodynamically stable hairpin struc ture. Its correct folding requires the presence of a ribozyme 3'-terminal s equence and the formation of helix P2. This confirms the ribozyme structure of the pseudoknot type and points to the crucial role of helix P2 in its o verall folding. Moreover, we show that the J4/2 region can be specifically cleaved in the presence of selected divalent metal ions in the full-length transcript, but not in a shorter one lacking six 3'-terminal nucleotides, w hich cannot form the pseudoknotted structure. Thus, a particular RNA confor mation around that cleavage site is required for specific hydrolysis, and t he J4/2 region seems to be involved in the formation of a general metal ion binding site. Recently, it has been proposed that, in the antigenomic ribo zyme, a four nucleotide sequence within the J1/2 region may contribute to t he folding pathway, being part of a mechanism responsible for controlling r ibozyme cleavage activity. Our study shows that in the genomic ribozyme the central hairpin region may contribute to a similar mechanism, providing a barrier to the formation of an active structure in the ribozyme folding pat hway. (C) 1999 Academic Press.