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 Sca I catalyzed ligation reaction between exogenously added oligomers: cucu + UCGa L-21 Sca I cucua + UCG. Steady-state kinetics for the forward and reverse direction were measured at 15 degrees C to find oligonucleotides that exhibit Michaelis-Menten behavior with acceptable KMS. 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.
Bevilacqua
P C
PC
Department of Chemistry, University of Rochester, New York 14627-0216, USA.
Sugimoto
N
N
Turner
D H
DH
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D005091:Exons; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D008954:Models, Biological; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation / Q000187:drug effects; D012326:RNA Splicing / Q000502:physiology*; D016337:RNA, Catalytic / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016053:RNA, Protozoan / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016808:Tetrahymena thermophila / Q000235:genetics* / Q000378:metabolism*
D016337:RNA, Catalytic; D016053:RNA, Protozoan; D008274:Magnesium