Comparison of pH Dependencies of the Tetrahymena Ribozyme Reactions with RNA 2'-Substituted and Phosphorothioate Substrates Reveals a Rate-Limiting Conformational Step

Herschlag, Daniel; Khosla, Mala

doi:10.1021/bi00183a036

Cited by 77 publications

(133 citation statements)

References 43 publications

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“…S and P with a 2′-deoxyribose substitution at position -1 were used to ensure that the chemical step was rate-limiting (57). This conclusion was confirmed by the observation of a log-linear pH dependence between pH 6.2 and 8.1 (data not shown, (5,59,60)). …”

Section: Measurement Of the Rate Constant For The Chemical Step And Tsupporting

confidence: 59%

Probing the Role of a Secondary Structure Element at the 5‘- and 3‘-Splice Sites in Group I Intron Self-Splicing: The Tetrahymena L-16 ScaI Ribozyme Reveals a New Role of the G·U Pair in Self-Splicing

2007

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Several ribozyme constructs have been used to dissect aspects of the group I self-splicing reaction. The Tetrahymena L-21 ScaI ribozyme, the best studied of these intron analogs, catalyzes a reaction analogous to the first step of self-splicing, in which a 5′-splice site analog (S) and guanosine (G) are converted into a 5′-exon analog (P) and GA. This ribozyme preserves the active site but lacks a short 5′-terminal segment (called the IGS extension herein) that forms dynamic helices, called the P1 extension and P10 helix. The P1 extension forms at the 5′-splice site in the first step of self-splicing and P10 forms at the 3′-splice site in the second step of self-splicing. To dissect the contributions from the IGS extension and the helices it forms we have investigated the effects of each of these elements at each reaction step. These experiments were performed with the L-16 ScaI ribozyme, which retains the IGS extension, and with 5′-and 3′-splice site analogs that differ in their ability to form the helices. The presence of the IGS extension strengthens binding of P by 40-fold, even when no new base pairs are formed. This large effect was especially surprising, as binding of S is essentially unaffected for S analogs that do not form additional base pairs with the IGS extension. Analysis of a U•U pair immediately 3′ to the cleavage site suggests that a previously identified deleterious effect from a dangling U residue on the L-21 ScaI ribozyme arises from a fortuitous active site interaction and has implications for RNA tertiary structure specificity. Comparisons of the affinities of 5′-splice site analogs that form only a subset of base pairs reveal that inclusion of the conserved G•U base pair at the cleavage site of group I introns destabilizes the P1 extension >100-fold relative to the stability of a helix with all Watson-Crick base pairs. Previous structural data with model duplexes and the recent intron structures suggest that this effect can be attributed to partial unstacking of the P1 extension at the G•U step. These results suggest a previously unrecognized role of the G•U wobble pair in self-splicing: breaking cooperativity in base pair formation between P1 and the P1 extensions. This effect may facilitate replacement of the P1 extension with P10 after the first chemical step of self-splicing and release of the ligated exons after the second step of self-splicing.The group I intron from Tetrahymena thermophila, the first catalytic RNA to be discovered, folds into a specific structure and performs a self-splicing reaction that includes chemical steps as well as RNA conformational rearrangements. This reaction is simple, relative to pre-mRNA † This work was supported by NIH grant GM49243. K.K. was supported in part by a predoctoral fellowship from the Boehringer Ingelheim NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript splicing and other RNA-mediated processes, and may serve as a tractable system to learn more about RNA catalysis and its functional conformational change...

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Section: Measurement Of the Rate Constant For The Chemical Step And Tsupporting

confidence: 59%

Probing the Role of a Secondary Structure Element at the 5‘- and 3‘-Splice Sites in Group I Intron Self-Splicing: The Tetrahymena L-16 ScaI Ribozyme Reveals a New Role of the G·U Pair in Self-Splicing

2007

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“…Previous work with E ScaI showed that the chemical step is pH-dependent, increasing in a log-linear fashion from pH ∼5-9 for -1d,rS (Herschlag & Khosla, 1994;Knitt & Herschlag, 1996). For the G414-containing ribozyme, the rate constant for approach to the internal equilibrium (k obs ) k f + k r ; Scheme 1) was also log-FIGURE 4: Regeneration of S* provides evidence for the establishment of the internal equilibrium (K int ).…”

Section: Observation Of An Internal Equilibrium For the L-21g414 Ribomentioning

confidence: 90%

Mechanistic Investigations of a Ribozyme Derived from the Tetrahymena Group I Intron: Insights into Catalysis and the Second Step of Self-Splicing

Mei

Herschlag

1996

Biochemistry

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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-21Scal 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 ∼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.Self-splicing of the Tetrahymena pre-rRNA proceeds in two consecutive phosphoryl transesterification steps ( Figure 1A; Cech, 1990). In the first step, an exogenous guanosine (G) 1 acting as a nucleophile attacks the 5′ splice site, leaving the 5′ exon with a 3′-hydroxyl terminus. In the second step, the 3′-terminal residue of the intron, G414, binds to the active site, and its 3′ phosphoryl group is attacked by the 3′-hydroxyl of the 5′ exon. This gives ligated exons and the free intron.The first step of self-splicing has been studied by using a shortened version of the intron. This version, referred to as the L-21ScaI ribozyme ( Figure 2B), has the first 21 and last 5 nucleotides removed and mimics the first step of the selfsplicing reaction ( Figure 1B). Extensive studies of the L-21ScaI ribozyme have provided insights into the mechanism of RNA catalysis and the first step of self-splicing (Cech et al., 1992).The second step of self-splicing, however, has not been characterized in as much detail. Several components have been shown to be involved in this step (Figure 2A). Guanosine is universally conserved a...

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“…An initial observation of a pK,, of -7 for the ribozyme reaction was consistent with general base catalysis by a ribozyme functional group with a perturbed pK,,. However, this observed pK, was shown to arise from a change from ratelimiting chemistry below pH 7 to a rate-limiting conformation step above pH 7 (Herschlag and Khosla 1994). These and additional pH studies indicate that a proton is lost prior to the chemical step from a group with pK, > 8, and the simplest candidate for this proton is the 3' OH of G (Herschlag et al 1993a;Herschlag andKhosla 1994: Knitt andHerschlag 1996).…”

Section: Phylogenetic Chemistrymentioning

confidence: 96%

“…Guanosine binding is unordered with respect to binding of S (Herschlag and Cech 1990a), although there is modest (-1 kcal/mol) energetic coupling between the two (Bevilacqua et al 1993;McConnell et al 1993). Once the ternary complex (EaG-S) is formed, transesterification occurs very rapidly (-300min-1 at 50 "C or 80min-' at 30°C; Herschlag andCech 1990a: McConnell et al 1993;Herschlag and Khosla 1994). Release of the 5'-product fragment, the one held onto the ribozyme by base-pairing and tertiary interactions.…”

Section: -Sequential Steps Of Ribozyme-catalyzed Rna Cleavagementioning

confidence: 99%

“…Finally. the reaction rate increases with p H between pH -5 and 7 and then levels off above pH 7, indicative of a rate-limiting conformational change (Herschlag and Khosla 1994). Thus these two group I ribozymes, differing in the identity of many of their active site nucleotides, are nevertheless functionally very similar (Zaug et al 1994).…”

Section: Phylogenetic Chemistrymentioning

confidence: 99%

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Group I Ribozymes: Substrate Recognition, Catalytic Strategies, and Comparative Mechanistic Analysis

Cech

Herschlag

1996

Nucleic Acids and Molecular Biology

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Comparison of pH Dependencies of the Tetrahymena Ribozyme Reactions with RNA 2'-Substituted and Phosphorothioate Substrates Reveals a Rate-Limiting Conformational Step

Cited by 77 publications

References 43 publications

Probing the Role of a Secondary Structure Element at the 5‘- and 3‘-Splice Sites in Group I Intron Self-Splicing: The Tetrahymena L-16 ScaI Ribozyme Reveals a New Role of the G·U Pair in Self-Splicing

Probing the Role of a Secondary Structure Element at the 5‘- and 3‘-Splice Sites in Group I Intron Self-Splicing: The Tetrahymena L-16 ScaI Ribozyme Reveals a New Role of the G·U Pair in Self-Splicing

Mechanistic Investigations of a Ribozyme Derived from the Tetrahymena Group I Intron: Insights into Catalysis and the Second Step of Self-Splicing

Group I Ribozymes: Substrate Recognition, Catalytic Strategies, and Comparative Mechanistic Analysis

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