STRUCTURAL AND KINETIC CHARACTERIZATION OF AN ACYL TRANSFERASE RIBOZYME

We have previously isolated, by in vitro selection, an acyl-transferas e ribozyme that is capable of transferring a biotinylated methionyl gr oup from the 3' end of a hexanucleotide substrate to its own 5'-hydrox yl. Comparison of the sequences of a family of evolved derivatives of this ribozyme allowed us to generate a model of the secondary structur e of the ribozyme. The predicted secondary structure was extensively t ested and confirmed by single-mutant and compensatory double-mutant an alyses. The role of the template domain in aligning the acyl-donor oli gonucleotide and acyl-acceptor region of the ribozyme was confirmed in a similar manner. The significance of different domains of the ribozy me structure and the importance of two tandem G:U wobble base pairs in the template domain were studied by kinetic characterization of mutan t ribozymes. The wobble base pairs contribute to the catalytic rate en hancement, but only in the context of the complete ribozyme; the riboz yme in turn alters the metal binding properties of this site. Competit ive inhibition experiments with unacylated substrate oligonucleotide a re consistent with the ribozyme acting to stabilize substrate binding to the template, while negative interactions with the aminoacyl portio n of the substrate destabilize binding.