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Mechanistic investigations of a ribozyme derived from the Tetrahymena group I intron: insights into catalysis and the second step of self-splicing.
Biochemistry. 1996 May 07; 35(18):5796-809.B

Abstract

Self-splicing of Tetrahymena pre-rRNA proceeds in two consecutive phosphoryl transesterification steps. One major difference between these steps is that in the first an exogenous guanosine (G) binds to the active site, while in the second the 3'-terminal G414 residue of the intron binds. The first step has been extensively characterized in studies of the L-21ScaI ribozyme, which uses exogenous G as a nucleophile. In this study, mechanistic features involved in the second step are investigated by using the L-21G414 ribozyme. The L-21G414 reaction has been studied in both directions, with G414 acting as a leaving group in the second step and a nucleophile in its reverse. The rate constant of chemical step is the same with exogenous G bound to the L-21ScaI ribozyme and with the intramolecular guanosine residue of the L-21G414 ribozyme. The result supports the previously proposed single G-binding site model and further suggests that the orientation of the bound G and the overall active site structure is the same in both steps of the splicing reaction. An evolutionary rationale for the use of exogenous G in the first step is also presented. The results suggest that the L-21G414 ribozyme exists predominantly with the 3'-terminal G414 docked into the G-binding site. This docking is destabilized by approximately 100-fold when G414 is attached to an electron-withdrawing pA group. The internal equilibrium with K(int) = 0.7 for the ribozyme reaction indicates that bound substrate and product are thermodynamically matched and is consistent with a degree of symmetry within the active site. These observations are consistent with the presence of a second Mg ion in the active site. Finally, the slow dissociation of a 5' exon analog relative to a ligated exon analog from the L-21G414 ribozyme suggests a kinetic mechanism for ensuring efficient ligation of exons and raises new questions about the overall self-splicing reaction.

Authors+Show Affiliations

Department of Biochemistry, Beckman Center B400, Stanford University, California 94305-5307, USA.No affiliation info available

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't
Research Support, U.S. Gov't, P.H.S.

Language

eng

PubMed ID

8639540

Citation

Mei, R, and D Herschlag. "Mechanistic Investigations of a Ribozyme Derived From the Tetrahymena Group I Intron: Insights Into Catalysis and the Second Step of Self-splicing." Biochemistry, vol. 35, no. 18, 1996, pp. 5796-809.
Mei R, Herschlag D. Mechanistic investigations of a ribozyme derived from the Tetrahymena group I intron: insights into catalysis and the second step of self-splicing. Biochemistry. 1996;35(18):5796-809.
Mei, R., & Herschlag, D. (1996). Mechanistic investigations of a ribozyme derived from the Tetrahymena group I intron: insights into catalysis and the second step of self-splicing. Biochemistry, 35(18), 5796-809.
Mei R, Herschlag D. Mechanistic Investigations of a Ribozyme Derived From the Tetrahymena Group I Intron: Insights Into Catalysis and the Second Step of Self-splicing. Biochemistry. 1996 May 7;35(18):5796-809. PubMed PMID: 8639540.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Mechanistic investigations of a ribozyme derived from the Tetrahymena group I intron: insights into catalysis and the second step of self-splicing. AU - Mei,R, AU - Herschlag,D, PY - 1996/5/7/pubmed PY - 1996/5/7/medline PY - 1996/5/7/entrez SP - 5796 EP - 809 JF - Biochemistry JO - Biochemistry VL - 35 IS - 18 N2 - Self-splicing of Tetrahymena pre-rRNA proceeds in two consecutive phosphoryl transesterification steps. One major difference between these steps is that in the first an exogenous guanosine (G) binds to the active site, while in the second the 3'-terminal G414 residue of the intron binds. The first step has been extensively characterized in studies of the L-21ScaI ribozyme, which uses exogenous G as a nucleophile. In this study, mechanistic features involved in the second step are investigated by using the L-21G414 ribozyme. The L-21G414 reaction has been studied in both directions, with G414 acting as a leaving group in the second step and a nucleophile in its reverse. The rate constant of chemical step is the same with exogenous G bound to the L-21ScaI ribozyme and with the intramolecular guanosine residue of the L-21G414 ribozyme. The result supports the previously proposed single G-binding site model and further suggests that the orientation of the bound G and the overall active site structure is the same in both steps of the splicing reaction. An evolutionary rationale for the use of exogenous G in the first step is also presented. The results suggest that the L-21G414 ribozyme exists predominantly with the 3'-terminal G414 docked into the G-binding site. This docking is destabilized by approximately 100-fold when G414 is attached to an electron-withdrawing pA group. The internal equilibrium with K(int) = 0.7 for the ribozyme reaction indicates that bound substrate and product are thermodynamically matched and is consistent with a degree of symmetry within the active site. These observations are consistent with the presence of a second Mg ion in the active site. Finally, the slow dissociation of a 5' exon analog relative to a ligated exon analog from the L-21G414 ribozyme suggests a kinetic mechanism for ensuring efficient ligation of exons and raises new questions about the overall self-splicing reaction. SN - 0006-2960 UR - https://www.unboundmedicine.com/medline/citation/8639540/Mechanistic_investigations_of_a_ribozyme_derived_from_the_Tetrahymena_group_I_intron:_insights_into_catalysis_and_the_second_step_of_self_splicing_ L2 - https://doi.org/10.1021/bi9527653 DB - PRIME DP - Unbound Medicine ER -