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.