Probing the interplay between the two steps of group I intron splicing: Competition of exogenous guanosine with omega G

One largely unexplored question about group I intron splicing is how the cl eavage and ligation steps of the reaction are coordinated. We describe a si mple in vitro trans-splicing model system in which both steps take place, i ncluding the exchange of ligands in the guanosine-binding site that must oc cur between the two steps. Using this model system, we show that the switch is accomplished by modulating the relative affinity of the binding site fu r the two ligands, While the terminal guanosine of the intron (omega G) and exogenous guanosine compete for binding during the first step of splicing, no competition is apparent during the second step, when omega G is bound t ightly. These results help explain how the ribozyme orchestrates progressio n through the splicing reaction. In addition to providing a new tool to ask basic questions about RNA catalysis, the trans-splicing model system will also facilitate the development of therapeutically useful group I ribozymes that can repair mutant mRNAs.