A SYNTHETIC MODEL FOR TRIPLE-HELICAL DOMAINS IN SELF-SPLICING GROUP-IINTRONS STUDIED BY ULTRAVIOLET AND CIRCULAR-DICHROISM SPECTROSCOPY

Structural studies were performed on synthetic oligonucleotides with s equences corresponding to the P4/P6 and J3/4, J6/7 regions of the self -splicing group I intron of the bacteriophage T4 nrdB pre-mRNA, which correspond to the proposed triple-helical domain in the Tetrahymena th ermophila intron. A 23-mer RNA was synthesized as a mixed ribo-deoxyri bo oligonucleotide, modeling an expected base-paired region P4 along w ith the J3/4 and P6 (5'-end bases of P6) regions. A third strand model ing the 3'-end bases of P6 and J6/7 regions, with which a triple helix may form, was synthesized as a pure oligoribonucleotide (7-mer RNA). The interactions of these oligonucleotides have been characterized by UV and circular dichroism (CD) spectroscopy. The results show that the 23-mer RNA forms a stable hairpin modeling the P4 base-paired region. Triple helix association between the 23-mer RNA hairpin and the 7-mer RNA single strand was detected by CD in the presence of Mg2+ (>5 mM) but not in presence of a monovalent cation like Na+ (up to 500 mM). St udies on selected variants of both 7-mer and 23-mer RNAs were carried out. The results show that for the association of the two partner stra nds not only the formation of P6 helix but also triplet interactions b etween the two strands are required. The association of the two strand s in general follow a pattern predicted by comparative sequence analys is. Parallel studies on pure oligodeoxyribonucleotides having base seq uences corresponding to those of the oligoribonucleotides showed no ev idence of association under similar conditions, which could indicate t hat the 2'-hydroxyl groups of the riboses might play an important role in hydrogen bonding to form the required nucleoside triples. Molecula r modeling studies on the proposed ''plaited triple helix'' formed by the association of the 23-mer RNA hairpin and 7-mer RNA single strand showed that the structure is sterically and energetically feasible.