Modification of Primary Structures of Hairpin Ribozymes for Probing Active Conformations

• Published in: Journal of molecular biology Vol. 252; no. 3; pp. 296 - 304
• Main Authors: Komatsu, Yasuo, Kanzaki, Ikuyo, Koizumi, Makoto, Ohtsuka, Eiko
• Format: Journal Article
• Language: English
• Published: England Elsevier Ltd 22-09-1995
• Subjects: Base Sequence; catalytic RNA; domain realignment; Hydrogen Bonding; Kinetics; Molecular Sequence Data; Nucleic Acid Conformation; ribozyme; RNA - chemical synthesis; RNA - chemistry; RNA, Catalytic - chemistry; RNA, Catalytic - metabolism; Structure-Activity Relationship; tertiary structure; domain realignment; tertiary structure; catalytic RNA; ribozyme
• ISSN: 0022-2836; 1089-8638
• DOI: 10.1006/jmbi.1995.0497

Hairpin ribozymes consist of two stem-loop domains, and these domains are assumed to interact with each other to produce the self-cleavage activity. We have studied the relationship of the tertiary structure of the hairpin ribozyme and the cleavage activity by dividing and re-joining the domains. A hairpin ribozyme (E50) was divided at the hinge region, and the main part was joined to a substrate (S1) using tri-or penta-cytidylates. These ribosymes retained the cleavage activity in the presence of the rest of the molecule, indicating that the active conformation could be maintained if the two domains interacted with each other. Based on the these results, we designed a new type of hairpin ribozyme by replacing one of the domains. To maintain the interaction of the domains, oligocytidylates were inserted at a junction. These reversely joined ribozyme complexes showed cleavage activity that was dependent on the linker lengths. These modifications in the primary structure of the hairpin ribozyme confirm the structural requirements for the catalytic reaction and provide information for the correlation of the tertiary structure with the cleavage of the hairpin ribozyme.