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

Abstract

Genomic RNA of the hepatitis delta agent has a highly conserved element of local tertiary structure. This element contains two nucleotides which become covalently crosslinked to each other upon irradiation with UV light. Using direct RNA analysis, we now identify the two nucleotides as U-712 and U-865 and show that the UV-induced crosslink can be broken 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 permit crosslinking. This model needs to be modified. Our data indicate that bases U-712 and U-865 closely approximate each other and suggest that the smooth helical contour proposed for delta RNA is interrupted by the UV-sensitive element. The nucleotide sequence shows that the UV-sensitive site does not have a particularly high density of conventional 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 RNase T1 at 37 degrees C for 45 min in 10 mM Tris-HCl, 1 mM EDTA (conditions expected to give complete digestion), this element can be isolated 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 complex.

Evidence for the Existence of the Loop E Motif of Potato Spindle Tuber Viroid In Vivo

“…UV cross-linking has been used for detecting tertiary interactions in vitro in a wide range of RNAs, for example, hepatitis delta virus (5,7,9), tRNAs (4), small nuclear RNAs (32), hairpin ribozymes (8), ribosomal RNAs (22), and Peach latent mosaic viroid (16). As shown here, this approach may be extended to investigate the in vivo existence of these tertiary interactions.…”

Hepatitis Delta Virus

“…Such information will have impact on such questions as RNA editing and RNA-directed transcription. Currently, all we know are the predicted rod-like structure, the ribozyme folding, and an unusual site that can be cross-linked with UV [13].…”

3-D models of the antigenomic ribozyme of the hepatitis delta agent with eight new contacts suggested by sequence analysis of 188 cDNA clones

“…Within these 25 bases, only a single mutation (at C-13) has been reported in sequenced isolates of the delta agent (41). During the replication cycle, the ribozyme sequences are constrained by factors in addition to those directly related to their catalytic capability; such as their ability to function as templates for replication, which may impose sequence restrictions (42); ability to form the rodlike structure (9); and their ability to generate the UV-sensitive tertiary structure (43,44) proposed as a control element for the genomic ribozyme (45,46). In our current in vitro study, in which none of these factors had an effect, the ranking of nucleotides from the least conserved to the most conserved was as follows: (the number of clones with a mutation at a particular position is given in parenthesis): G (47), nucleotides contributing to tertiary structure are known to be highly conserved.…”