A mechanistic framework for co-transcriptional folding of the HDV genomic ribozyme in the presence of downstream sequence.

Hepatitis delta virus (HDV) is a circular pathogenic RNA that uses self-cleavage by closely related 84 nt genomic and antigenomic ribozymes to facilitate the replication of its genome. Downstream of each ribozyme is a stretch of nucleotides termed the attenuator that functions to base-pair with and unfold the ribozyme into a rod-like fold. The competing rates of RNA synthesis, ribozyme folding and cleavage, and rod folding are therefore likely to affect the efficiency of co-transcriptional self-cleavage. In these studies, co-transcriptional folding of the genomic ribozyme was assayed in vitro by monitoring co-transcriptional self-cleavage of transcripts having variable lengths of sequence downstream of the ribozyme. Co-transcriptional cleavage data were simulated successfully only with kinetic models in which cleavage-inactive channels were populated during transcription. Partitioning to and escape from these channels was influenced, in part, by whether the available attenuator sequence could form structures with the ribozyme, and by the stability of such structures. Surprisingly, only 23 nt of attenuator were needed for strong inactivation of cleavage. Self-cleavage of certain 3'-virus-containing sequences could be restored, partially, by renaturation; however, self-cleavage of transcripts with a full-length attenuator could not be restored efficiently by renaturation in vitro. This suggests that in the presence of the attenuator, the cleavage-active ribozyme fold is not the thermodynamically most stable species. In accordance with this model, the efficiency of self-cleavage of the ribozyme followed by a full-length attenuator was increased by decreasing the rate of transcription. These results suggest that, in the absence of additional factors, efficient co-transcriptional cleavage of the full-length genomic HDV RNA may require cleavage to occur prior to synthesis of the attenuator.