A mechanistic framework for the second step of splicing catalyzed by the Tetrahymena ribozyme

A simple model system is described which mimics the second step of splicing and reverse cyclization reactions of the self-splicing intron from Tetrahymena thermophila. This model is based on the L-21 Sea I catalyzed ligation reaction between exogenously added oligomers: cucu + UCGa cucua + UCG. Steady-state kinetics for the forward and reverse direction were measured at 15 °C to find oligonucleotides that exhibit Michaelis-Menten behavior with acceptable R(M)s. CUCU and UCGA fit both criteria and were chosen for further studies. Steady-state kinetics reveal a lag that appears to be an RNA folding step that is eliminated by preincubation of the ribozyme with 2 mM and higher [Mg²⁺] and by UCGA. At constant ionic strength, the Mg²⁺ dependence of steady state rates exhibits a sharp maximum near 5 mM Mg²⁺. Pre-steady-state and steady-state kinetics, along with active-site titrations, explain the Mg²⁺ profile: the rate of reaction up to and including chemistry increases with Mg²⁺ concentration, while the fraction of active ribozyme and the rate of postchemistry steps decrease with Mg²⁺ concentration. The rate-limiting step at 5 mM Mg²⁺ for the reaction mimicking the second step of splicing is either chemistry or a conformational change before chemistry involving ribozyme bound with substrates. The rate- limiting step at 50 mM Mg²⁺ appears to be a postchemistry conformational change of the ribozyme or product release. At 50 mM Mg²⁺, single-turnover experiments support ordered binding of substrates with 5'-exon mimic binding before 3'-splice site mimic. Moreover, the 3'-splice site mimic binds and reacts in the presence of 5'-exon mimics predocked into the catalytic core. Results also indicate that Mg²⁺ ions associate with the ribozyme upon docking.