Tm studies of a tertiary structure from the human hepatitis delta agent which functions in vitro as a ribozyme control element.

Viroids and other circular subviral RNA pathogens, such as the hepatitis delta agent, use a rolling circle replication cycle requiring an intact circular RNA. However, many infectious RNAs have the potential to form self-cleavage structures, whose formation must be controlled in order to preserve the circular replication template. The native structure of delta RNA contains a highly conserved element of local tertiary structure which is composed of sequences partially overlapping those needed to form the self-cleavage motif. A bimolecular complex containing the tertiary structure can be made. We show that when it is part of this bimolecular complex the potential cleavage site is protected and is not cleaved by the delta ribozyme, demonstrating that the element of local tertiary structure can function as a ribozyme control element in vitro. Physical studies of the complex containing this element were carried out. The complex binds magnesium ions and is not readily dissociated by EDTA under the conditions tested; > 50% of the complexes remain following incubation in 1 mM EDTA at 60 degrees C for 81 min. The thermal stability of the complex is reduced in the presence of sodium ions. A DNA complex and a perfect RNA duplex studied in parallel showed a similar effect, but of lesser magnitude. The RNA complex melts at temperatures approximately 10 degrees C lower in buffers containing 0.5 mM MgCl2 and 100 mM NaCl than in buffers containing 0.5 mM MgCl2 with no NaCl (78.1 compared with 87.7 degrees C). The element of local tertiary structure in delta genomic RNA appears to be a molecular clamp whose stability is highly sensitive to ion concentration in the physiological range.

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

We mapped 359 mutations at 25 positions in synthetic variants of the antigenomic ribozyme of the hepatitis delta agent by analyzing the sequences of 188 cDNA clones. These data were used to identify three features of the ribozyme: highly conserved nucleotides, positions with restricted nucleotide substitutions and three-dimensional relationships between nucleotides. The distribution of mutations at the 25 positions was as follows: G-11 (the eleventh nucleotide from the cleavage site) was mutated in 56 clones; G-12 in 36; U-15 in 33; C-13 in 26; G-28 in 23; C-27 in 21; C-29 in 19; U-26 in 17; C-18 in 14; A-14 in 13; C-16 in 13; C-19 in 12; U-17 in 11; A-20 in 10; G-42 in 9; G-40 in 7; G-41 in 7; C-24 in 6; U-32 in 6; U-23 in 5; C-25 in 4; C-21 in 3; G-30 in 3; G-31 in 3; C-22 in 1. All clones containing a mutation at C-25 had an A at this position, suggesting that the extra cyclic amino group present in adenine and cytosine may function during the cleavage event. Mutations at certain positions were common in simple clones (containing only one or two mutations), while mutations at other positions were over-represented in more complex clones. Both compensatory base changes and co-mutational frequencies were used to identify eight pairs of nucleotides which may interact with each other: G-11 and C-18, G-12 and C-27, C-13 and G-28, C-21 and U-23/C-24, C-21 and G-30, U-23 and G-31/U-32, C24 and G-30, C-27 and G-42. These pairs, which involve some of the most conserved positions in the molecule, suggest interactions among nucleotides previously depicted in open-loop structures. The newly proposed points of contact between pairs of nucleotides are compatible with both the axehead and pseudoknot secondary structural models and were combined with previously proposed Watson-Crick base paired helices to produce two three dimensional models. In both of these, C-25 and C-76 are placed near the cleavage site.

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 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.