NOVEL SYSTEM FOR ANALYSIS OF GROUP-I 3'-SPLICE-SITE REACTIONS BASED ON FUNCTIONAL TRANS-INTERACTION OF THE P1 P10 REACTION HELIX WITH THE RIBOZYMES CATALYTIC CORE/

A group I intron from a bacterial tRNA precursor has been converted in to an RNA enzyme that catalyzes the efficient polymerization of oligor ibonucleotide analogs of tRNA exons using a reaction scheme consisting of multiple cycles of reverse and forward exon ligation reactions. He re, we present results showing that this system represents a novel and useful tool for the analysis of 3' splice site reactions of group I r ibozymes. First, analysis of variant substrates containing base substi tutions in group I secondary structure; elements P1, P9.0 and P10 conf irms that exon polymerization is dependent on these structures, and th erefore constitutes an appropriate and relevant model system for study ing the exon ligation step of splicing. Second, to probe interactions between the intron's catalytic core and the bases and backbone of the P1/P10 reaction helix, two successful strategies for separating the in ternal guide sequence from the intron core were devised. One such stra tegy uses a construct in which the reaction helix interacts functional ly with the catalytic core using only tertiary contacts. Further stabi lization of this interaction through the inclusion of a 7 bp intermole cular P2 helix generates increased reaction efficiency. Third, when pr ovided with two reaction helices, the ribozyme synthesizes mixed polym ers through a mechanism that involves sequential binding and release o f the duplexes. Fourth, in these reactions, turnover of the external g uide sequence requires unwinding and annealing of the P2 helix, sugges ting that P2 unwinding may occur during group I splicing. These result s provide novel experimental tools to probe the relatively poorly unde rstood 3' splice site reactions of group I introns, and may be relevan t to ribozyme-catalyzed assembly and recombination of oligomers in pre biotic scenarios.