title: Structural and kinetic characterization of an acyl transferase ribozyme.
abstract: We have previously isolated, by in vitro selection, an acyl-transferase ribozyme that is capable of transferring a biotinylated methionyl group from the 3' end of a hexanucleotide substrate to its own 5'-hydroxyl. Comparison of the sequences of a family of evolved derivatives of this ribozyme allowed us to generate a model of the secondary structure of the ribozyme. The predicted secondary structure was extensively tested and confirmed by single-mutant and compensatory double-mutant analyses. The role of the template domain in aligning the acyl-donor oligonucleotide and acyl-acceptor region of the ribozyme was confirmed in a similar manner. The significance of different domains of the ribozyme structure and the importance of two tandem G:U wobble base pairs in the template domain were studied by kinetic characterization of mutant ribozymes. The wobble base pairs contribute to the catalytic rate enhancement, but only in the context of the complete ribozyme; the ribozyme in turn alters the metal binding properties of this site. Competitive inhibition experiments with unacylated substrate oligonucleotide are consistent with the ribozyme acting to stabilize substrate binding to the template, while negative interactions with the aminoacyl portion of the substrate destabilize binding.
authors.affiliation: Department of Molecular Biology, Massachusetts General Hospital, Boston 02114, USA.
authors.forename: H
authors.initials: H
authors.lastname: Suga
authors.forename: P A
authors.initials: PA
authors.lastname: Lohse
authors.forename: J W
authors.initials: JW
authors.lastname: Szostak
mesh_terms: D000217:Acyltransferases / Q000737:chemistry*; D001482:Base Composition; D001483:Base Sequence; D019921:Biotinylation; D019020:Directed Molecular Evolution; D008958:Models, Molecular; D015394:Molecular Structure; D009154:Mutation; D009841:Oligonucleotides / Q000737:chemistry; D016133:Polymerase Chain Reaction; D016337:RNA, Catalytic / Q000737:chemistry* / Q000235:genetics; D017421:Sequence Analysis; D013698:Templates, Genetic
chemical_list: D009841:Oligonucleotides; D016337:RNA, Catalytic; D000217:Acyltransferases