title: The loop B domain is physically separable from the loop A domain in the hairpin ribozyme.
abstract: In order to understand the catalysis mechanism of the hairpin ribozyme, mutant ribozymes were constructed. The distance between the loop A domain and the loop B domain was extended by inserting various lengths of nucleotide linkers at the hinge region in cis mutants, or the domains were separated physically in a trans mutant. All the mutant ribozymes, including the trans mutant, could cleave substrate RNA at the predicted site. A cis mutant with a single nucleotide insertion exhibited cleavage activity about twice as high as that of the wild-type (wt) ribozyme. The insertion of 2-5 nucleotides (nt) gradually reduced the activity to the level of the wt ribozyme. Insertion of a longer linker, up to 11 nt, resulted in the reduction of activity to one half of that of the wt ribozyme. The ribozyme with a single nucleotide insertion at the hinge region seems to form a more suitable conformation for catalysis by three-dimensional fold-back of the loop B to loop A containing the cleavage site. The trans mutant, in which the A and B domains were physically separated, maintained a significant level of activity, suggesting that both domains are necessary for catalysis, but separable. These results demonstrate that interaction between the A and B domains results in catalysis.
authors: Research Center for New BioMaterials in Agriculture, Seoul National University, Suwon, Korea.
authors: C
authors: C
authors: Shin
authors: J N
authors: JN
authors: Choi
authors: S I
authors: SI
authors: Song
authors: J T
authors: JT
authors: Song
authors: J H
authors: JH
authors: Ahn
authors: J S
authors: JS
authors: Lee
authors: Y D
authors: YD
authors: Choi
mesh_terms: D001483:Base Sequence; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D009690:Nucleic Acid Conformation; D016337:RNA, Catalytic / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity
chemical_list: D016337:RNA, Catalytic