Modular engineering of a Group I intron ribozyme

• Published in: Nucleic acids research 2002-08, Vol.30 (15), p.3473-3480
• Main Authors: Ohuchi, Shoji J., Ikawa, Yoshiya, Shiraishi, Hideaki, Inoue, Tan
• Format: Article
• Language: English
• Subjects: Base Sequence; Catalytic Domain; Consensus Sequence; DNA Mutational Analysis; Gene Library; Genetic Engineering - methods; Introns; Kinetics; Molecular Sequence Data; Nucleic Acid Conformation; Polymerase Chain Reaction; RNA, Catalytic - chemistry; RNA, Catalytic - genetics; RNA, Catalytic - metabolism
• ISSN: 0305-1048; 1362-4962; 1362-4962
• DOI: 10.1093/nar/gkf453

All Group I intron ribozymes contain a conserved core region consisting of two helical domains, P4–P6 and P3–P7. Recent studies have demonstrated that the elements required for catalysis are concentrated in the P3–P7 domain. We carried out in vitro selection experiments by using three newly constructed libraries on a variant of the T4 td Group I ribozyme containing only a P3–P7 domain in its core. Selected variants with new peripheral elements at L7.1, L8 or L9 after nine cycles efficiently catalyzed the reversal reaction of the first step of self‐splicing. The variants from this selection contained a short sequence complementary to the substrate RNA without exception. The most active variant, which was 3‐fold more active than the parental wild‐type ribozyme, was developed from the second selection by employing a clone from the first selection. The results show that the P3–P7 domain can stand as an independent catalytic module to which a variety of new domains for enhancing the activity of the ribozyme can be added.