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

Philip C. Bevilacqua, Naoki Sugimoto, Douglas H. Turner

Research output: Contribution to journalArticlepeer-review

28 Scopus citations


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.

Original languageEnglish (US)
Pages (from-to)648-658
Number of pages11
Issue number2
StatePublished - Jan 16 1996

All Science Journal Classification (ASJC) codes

  • Biochemistry


Dive into the research topics of 'A mechanistic framework for the second step of splicing catalyzed by the Tetrahymena ribozyme'. Together they form a unique fingerprint.

Cite this