Modular engineering of a Group I intron ribozyme.
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.
Ohuchi
Shoji J
SJ
Graduate School of Science and. Graduate School of Biostudies, Kyoto University, Kyoto 606-8502, Japan.
Ikawa
Yoshiya
Y
Shiraishi
Hideaki
H
Inoue
Tan
T
D001483:Base Sequence; D020134:Catalytic Domain; D016384:Consensus Sequence; D004252:DNA Mutational Analysis; D015723:Gene Library; D005818:Genetic Engineering / Q000379:methods*; D007438:Introns; D007700:Kinetics; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D016133:Polymerase Chain Reaction; D016337:RNA, Catalytic / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism
C116167:GIR1 ribozyme; D016337:RNA, Catalytic