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

Abstract

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...

Fitness landscape of a dynamic RNA structure

“…N 2 ..N 5 ). This supports earlier models, which argued that P1ex function is largely independent of sequence as long as minimal structural requirements (including avoidance of excess stability) are satisfied (Doudna et al 1989;Allain and Varani 1995;Karbstein et al 2007).…”

“…Although the presence of neither P1ex nor P10 is strictly required for splicing (Been and Cech 1985;Price and Cech 1988;Cech 1990), both helices contribute to splicing efficiency, as they facilitate splice site alignment and exon ligation and reduce non-productive alternative interactions, including the use of cryptic splice sites (Michel et al 1989;Suh and Waring 1990;Narlikar et al 2000;Bell et al 2004;Karbstein et al 2007). Mutations in P1ex and P10 have previously been shown to affect rates of catalysis at different stages of splicing (Doudna et al 1989;Guo and Cech 2002;Bell et al 2004;Karbstein et al 2007), which is relevant for knt production and, subsequently, fitness (Guo and Cech 2002). Prior work has also provided prima facie evidence for antagonistic pleiotropy, inferring from a small collection of individual mutants that overly stable pairing in P1ex might be selected against because it impedes dissociation and therefore P10 formation (Guo and Cech 2002;Michael A Bell et al 2004).…”

DEAD-box proteins can completely separate an RNA duplex using a single ATP

“…S4). The G⅐U pair has been shown to destabilize the P1 helix, relative to the G-C, in part by disrupting base stacking (24,25). We tested the effects of this base pair in the context of the 6-bp and 11-bp duplexes.…”

Probing the Dynamics of the P1 Helix within the Tetrahymena Group I Intron

“…Ribozymes were prepared by in vitro transcription with T7 RNA polymerase and purified by gel electrophoresis as previously described for the wild type and mutant L-16 ScaI ribozyme32 and for the L-21 ScaI ribozyme used in cross-linking experiments31. Lac repressor (LacI) was expressed from plasmid pMDB1 (a gift from Michael Brenowitz, Albert Einstein College of Medicine) in E. coli and was purified as described33.…”