AN RNA TERTIARY STRUCTURE OF THE HEPATITIS-DELTA AGENT CONTAINS UV-SENSITIVE BASES U-712 AND U-865 AND CAN FORM IN A BIMOLECULAR COMPLEX

Genomic RNA of the hepatitis delta agent has a highly conserved elemen t of local tertiary structure. This element contains two nucleotides w hich become covalently crosslinked to each other upon irradiation with UV light. Using direct RNA analysis, we now identify the two nucleoti des as U-712 and U-865 and show that the UV-induced crosslink can be b roken by re-exposure to a 254 nm peak UV light source. In the rod-like secondary structural model of delta RNA, nucleotides U-712 and U-865 are off-set from each other by 5-6 bases, a distance too great to perm it crosslinking. This model needs to be modified. Our data indicate th at bases U-712 and U-865 closely approximate each other and suggest th at the smooth helical contour proposed far delta RNA is interrupted by the UV-sensitive element. The nucleotide sequence shows that the UV-s ensitive site does not have a particularly high density of conventiona l Watson-Crick base pairs compared to the rest of the genome. However, this element may have a number of non-Watson-Crick bonds which confer stability. Following UV-crosslinking and digestion with 1 mg/ml of RN ase T1 at 37 degrees C for 45 min in 10 mM Tris-HCl, 1 mM EDTA (condit ions expected to give complete digestion), this element can be isolate d as part of a 54 nucleotide long partial digestion product containing at least 16 internal G residues. UV-crosslinking analysis shows that this unusual tertiary structural element can form in a bimolecular com plex.