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

Mei, Rui; Herschlag, Daniel

doi:10.1021/bi9527653

Cited by 50 publications

(80 citation statements)

References 44 publications

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“…The logarithm of the rate as a function of pH is a line with a slope of 0.9 ( Figure 3), indicating that a single deprotonation is rate limiting. Such behavior for ribozyme reactions involving phosphoryl transfer is taken as evidence of a ratelimiting chemical step, because the attacking hydroxyl must be deprotonated during this step (15,(17)(18)(19). Thus, the thio effect seen upon substitution of the pro-S P oxygen can be attributed solely to the elemental effect on the reactivity of the phosphate reaction center.…”

Section: Resultsmentioning

confidence: 99%

Recognition of Nucleoside Triphosphates during RNA-Catalyzed Primer Extension

et al. 2000

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In support of the idea that certain RNA molecules might be able to catalyze RNA replication, a ribozyme was previously generated that synthesizes short segments of RNA in a reaction modeled after that of proteinaceous RNA polymerases. Here, we describe substrate recognition by this polymerase ribozyme. Altering base or sugar moieties of the nucleoside triphosphate only moderately affects its utilization, provided that the alterations do not disrupt Watson-Crick pairing to the template. Correctly paired nucleotides have both a lower K m and a higher k cat , suggesting that differential binding and orientation each play roles in discriminating matched from mismatched nucleotides. Binding of the pyrophosphate leaving group appears weak, as evidenced by a very inefficient pyrophosphate-exchange reaction, the reverse of the primer-extension reaction. Indeed, substitutions at the γ-phosphate can be tolerated, although poorly. Thio substitutions of oxygen atoms at the reactive phosphate exert effects similar to those seen with cellular polymerases, leaving open the possibility of an active site analogous to those of protein enzymes. The polymerase ribozyme, derived from an efficient RNA ligase ribozyme, can achieve the very fast k cat of the parent ribozyme when the substrate of the polymerase (GTP) is replaced by an extended substrate (pppGGA), in which the GA dinucleotide extension corresponds to the second and third nucleotides of the ligase. This suggests that the GA dinucleotide, which had been deleted when converting the ligase into a polymerase, plays an important role in orienting the 5′-terminal nucleoside. Polymerase constructs that restore this missing orientation function should achieve much more efficient and perhaps more accurate RNA polymerization.The RNA world hypothesis proposes that early life relied on catalytic RNAs rather than protein enzymes for catalysis (1). Much of the appeal of this theory comes from the notion that ribozymes, which could have served as their own genes, would have been much simpler to duplicate than proteins (2-5). Hence, a vital component of the RNA world scenario is an RNA polymerase ribozyme responsible for replicating the essential ribozymes of early life (including itself). Whether there might be RNA sequences that can promote RNA polymerization with sufficient accuracy and efficiency for RNA self-replication is one of the fundamental unanswered questions related to the RNA world hypothesis (1, 6). Finding such a sequence would support the idea of the RNA world and provide a key component for the synthesis of simple living cells (7).The class I ligase ( Figure 1A) is a promising starting point for efforts to develop a self-replicating ribozyme. This ribozyme ligates two RNAs, generating a 3′,5′-phosphodiester linkage and releasing pyrophosphate, a reaction similar to that of RNA and DNA polymerases (8). Engineered forms of the ligase can polymerize short segments of RNA ( Figure 1C). In the presence of appropriate RNA templates and nucleoside triphosphates (NTP...

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Section: Resultsmentioning

confidence: 99%

Recognition of Nucleoside Triphosphates during RNA-Catalyzed Primer Extension

et al. 2000

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“…31,32 These data and direct measurements of substrate association 33 implied that P1 remains docked with J4/5 during the catalytic steps of splicing, even while undergoing molecular motions like the unwinding of the 5 0 end of the intron. Since the docking of P1 and J4/5 is maintained, we conclude that the main conformational changes between steps 1 and 2 are more likely to be centered around P9.0.…”

Section: Modelling Of the Architecturementioning

confidence: 86%

Architecture and folding mechanism of the Azoarcus Group I Pre-tRNA

Rangan

Masquida

Westhof

et al. 2004

Journal of Molecular Biology

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“…The details of the pre-steady-state kinetic methods employed are given in the Materials and Methods and follow directly from previous studies (1,5,6,23,35,36). We then present and compare the frameworks.…”

Section: Kinetic and Thermodynamic Framework For The L-16 Scai Ribozmentioning

confidence: 99%

“…The group I self-splicing reaction has been studied in the greatest depth with shortened ribozyme constructs derived from the self-splicing intron ( (1,5,8,20,21,30,(35)(36)(37), see also: (38,39)). The most commonly studied of these constructs is the L-21 ScaI ribozyme ( Figure 1B).…”

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confidence: 99%

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

Cited by 50 publications

References 44 publications

Recognition of Nucleoside Triphosphates during RNA-Catalyzed Primer Extension

Recognition of Nucleoside Triphosphates during RNA-Catalyzed Primer Extension

Architecture and folding mechanism of the Azoarcus Group I Pre-tRNA

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

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