3-D MODELS OF THE ANTIGENOMIC RIBOZYME OF THE HEPATITIS-DELTA AGENT WITH 8 NEW CONTACTS SUGGESTED BY SEQUENCE-ANALYSIS OF 188 CDNA CLONES

We mapped 359 mutations at 25 positions in synthetic variants of the a ntigenomic ribozyme of the hepatitis delta agent by analyzing the sequ ences of 188 cDNA clones, These data were used to identify three featu res of the ribozyme: highly conserved nucleotides, positions with rest ricted nucleotide substitutions and three-dimensional relationships be tween nucleotides. The distribution of mutations at the 25 positions w as as follows: G-11 (the eleventh nucleotide from the cleavage site) w as mutated in 56 clones; G-12 in 36; U-15 in 33; C-13 in 26; G-28 in 2 3; 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 prese nt in adenine and cytosine may function during the cleavage event. Mut ations at certain positions were common in simple clones (containing o nly one or two mutations), while mutations at other positions were ove r-represented in more complex clones. Both compensatory base changes a nd cc-mutational frequencies were used to identify eight pairs of nucl eotides 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 th e most conserved positions in the molecule, suggest interactions among nucleotides previously depicted in open-loop structures. The newly pr oposed points of contact between pairs of nucleotides are compatible w ith both the axehead and pseudoknot secondary structural models and we re 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.