A Mechanistic Framework for the Second Step of Splicing Catalyzed by the Tetrahymena Ribozyme
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- 作者：Philip C. Bevilacqua ; Naoki Sugimoto ; and Douglas H. Turner
- 刊名：Biochemistry
- 出版年：1996
- 出版时间：January 16, 1996
- 年：1996
- 卷：35
- 期：2
- 页码：648 - 658
- 全文大小：743K
- 年卷期：v.35,no.2(January 16, 1996)
- ISSN：1520-4995
文摘
A simple model system is described which mimics the second step ofsplicing and reverse cyclization reactions of the self-splicing intron fromTetrahymena thermophila. This model isbased on the L-21 Sca I catalyzed ligation reaction between exogenouslyadded oligomers: cucu +UCGa
bscribe/journals/bichaw/35/i02/eqn/bi951962ze10001.gif"> cucua + UCG. Steady-state kinetics for the forward and reversedirection weremeasured at 15 
C to find oligonucleotides that exhibitMichaelis-Menten behavior with acceptableKMs.CUCU and UCGA fit both criteria and were chosen for furtherstudies. Steady-state kinetics reveal a lagthat appears to be an RNA folding step that is eliminated bypreincubation of the ribozyme with 2 mMand higher [Mg2+] and by UCGA. At constant ionicstrength, the Mg2+ dependence of steady-stateratesexhibits a sharp maximum near 5 mM Mg2+.Pre-steady-state and steady-state kinetics, along withactive-site titrations, explain the Mg2+ profile: the rate ofreaction up to and including chemistry increases withMg2+ concentration, while the fraction of active ribozymeand the rate of postchemistry steps decreasewith Mg2+ concentration. The rate-limiting step at 5mM Mg2+ for the reaction mimicking thesecondstep of splicing is either chemistry or a conformational change beforechemistry involving ribozyme boundwith substrates. The rate-limiting step at 50 mMMg2+ appears to be a postchemistryconformationalchange of the ribozyme or product release. At 50 mMMg2+, single-turnover experiments supportorderedbinding of substrates with 5'-exon mimic binding before 3'-splice sitemimic. Moreover, the 3'-splicesite mimic binds and reacts in the presence of 5'-exon mimicspredocked into the catalytic core. Resultsalso indicate that Mg2+ ions associate with the ribozymeupon docking.
C to find oligonucleotides that exhibitMichaelis-Menten behavior with acceptableKMs.CUCU and UCGA fit both criteria and were chosen for furtherstudies. Steady-state kinetics reveal a lagthat appears to be an RNA folding step that is eliminated bypreincubation of the ribozyme with 2 mMand higher [Mg2+] and by UCGA. At constant ionicstrength, the Mg2+ dependence of steady-stateratesexhibits a sharp maximum near 5 mM Mg2+.Pre-steady-state and steady-state kinetics, along withactive-site titrations, explain the Mg2+ profile: the rate ofreaction up to and including chemistry increases withMg2+ concentration, while the fraction of active ribozymeand the rate of postchemistry steps decreasewith Mg2+ concentration. The rate-limiting step at 5mM Mg2+ for the reaction mimicking thesecondstep of splicing is either chemistry or a conformational change beforechemistry involving ribozyme boundwith substrates. The rate-limiting step at 50 mMMg2+ appears to be a postchemistryconformationalchange of the ribozyme or product release. At 50 mMMg2+, single-turnover experiments supportorderedbinding of substrates with 5'-exon mimic binding before 3'-splice sitemimic. Moreover, the 3'-splicesite mimic binds and reacts in the presence of 5'-exon mimicspredocked into the catalytic core. Resultsalso indicate that Mg2+ ions associate with the ribozymeupon docking.