Minimum secondary structure requirements for catalytic activity of a self-splicing group I intron

Beaudry, Amber A.; Joyce, Gerald F.

doi:10.1021/bi00479a027

Cited by 62 publications

(42 citation statements)

References 33 publications

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“…Deletion of the 3′-peripheral elements (P9.1 and P9.2) results in decreased protection of the conserved core from Fe(II)‚EDTA cleavage and in changes in RNase T1 protection in the IGS, as well as large decreases in intron self-splicing activity (30,31,70). Similarly, removal of the P2 and P2.1 elements results in reduced 5′-exon cleavage activity (27,71). This suggests that the 3′-peripheral elements and P2-P2.1 may, like P5abc, be important for cooperative folding to the active ribozyme structure, although measurement of individual reaction steps would be required to address this fully.…”

Section: Discussionmentioning

confidence: 99%

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The P5abc Peripheral Element Facilitates Preorganization of the Tetrahymena Group I Ribozyme for Catalysis

et al. 2000

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Phylogenetic comparisons and site-directed mutagenesis indicate that group I introns are composed of a catalytic core that is universally conserved and peripheral elements that are conserved only within intron subclasses. Despite this low overall conservation, peripheral elements are essential for efficient splicing of their parent introns. We have undertaken an in-depth structure-function analysis to investigate the role of one of these elements, P5abc, using the well-characterized ribozyme derived from the Tetrahymena group I intron. Structural comparisons using solution-based free radical cleavage revealed that a ribozyme lacking P5abc (E ∆P5abc ) and E ∆P5abc with P5abc added in trans (E ∆P5abc ‚P5abc) adopt a similar global tertiary structure at Mg 2+ concentrations greater than 20 mM [Doherty, E. A., et al. (1999) Biochemistry 38, 2982-90]. However, free E ∆P5abc is greatly compromised in overall oligonucleotide cleavage activity, even at Mg 2+ concentrations as high as 100 mM. Further characterization of E ∆P5abc via DMS modification revealed local structural differences at several positions in the conserved core that cluster around the substrate binding sites. Kinetic and thermodynamic dissection of individual reaction steps identified defects in binding of both substrates to E ∆P5abc , with g25-fold weaker binding of a guanosine nucleophile and g350-fold weaker docking of the oligonucleotide substrate into its tertiary interactions with the ribozyme core. These defects in binding of the substrates account for essentially all of the 10 4 -fold decrease in overall activity of the deletion mutant. Together, the structural and functional observations suggest that the P5abc peripheral element not only provides stability but also positions active site residues through indirect interactions, thereby preferentially stabilizing the active ribozyme structure relative to alternative less active states. This is consistent with the view that peripheral elements engage in a network of mutually reinforcing interactions that together ensure cooperative folding of the ribozyme to its active structure.Catalytic RNAs can provide rate enhancements that rival those of protein enzymes (1-3). To accomplish this, an RNA must contain sufficient information in its primary sequence to meet two challenges: it must be able to form stable tertiary structure, and it must be able to stabilize the active structure relative to alternate inactive and less active conformations. Relative to proteins, RNA is expected to encounter structural difficulties arising from the charged nature and greater rotational freedom of the phosphodiester backbone, the limited number of nucleoside bases, and the sequestration of the bases by base-pairing within secondary structure (4, 5). We have focused on group I introns to study the means by which RNA molecules can achieve functional structures.The wealth of functional and structural information available for group I introns makes them a powerful system in which to examine the structural underpin...

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Section: Discussionmentioning

confidence: 99%

“…The low overall conservation of peripheral element sequence and identity, their distance from the active site, and the increased Mg 2+ requirement for ribozyme folding and activity upon their deletion suggest these elements act indirectly on the active site by stabilizing and/or modulating the intron fold (24,25,27,(30)(31)(32).…”

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confidence: 99%

The P5abc Peripheral Element Facilitates Preorganization of the Tetrahymena Group I Ribozyme for Catalysis

et al. 2000

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“…However, the transposant W•Y•X•Z retained minimal activity as a consequence of the ability of RNA fragments to function in trans and provide multiple functions (Beaudry and Joyce, 1990;Vaidya and Lehman, 2009;van der Horst et al, 1991). (Figure 41), indicating the ability of a recombinase system to explore various sequences in a quasispecies cloud to increase local fitness.…”

Section: A Global Search For Functional Moleculesmentioning

confidence: 99%

“…Regardless, these 'exogenous' GUGs could potentially complement the missing canonical IGS if they were to work in trans. Such activity would be similar to the manner in which fragments of the Tetrahymena ribozyme can noncovalently assemble to restore activity, although with each RNA presumably only adopting one role (Beaudry and Joyce, 1990;van der Horst et al, 1991).…”

Section: A Group I Intron Without An Igsmentioning

confidence: 98%

“…Notably, these enzymes often remain active when broken into non-covalent trans complexes. The work of Inoue and others has shown that the Tetrahymena, sunY, Azoarcus, Synechococcus, phage T4 td and other group I introns can retain catalytic efficiency when fragmented into several non-covalent pieces and then reconstituted in trans via base-pairing and tertiary interactions (Beaudry and Joyce, 1990;Chowrira et al, 1995;Doudna and Cech, 1995;Ikawa et al, 2000a;van der Horst et al, 1991). These studies have isolated the regions of the ribozyme that are necessary to carry out the various catalytic events of self-splicing, a multi-step process that involves two transesterification reactions plus often a hydrolytic cleavage reaction.…”

Section: Introductionmentioning

confidence: 99%

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Spontaneous Cooperative Assembly of Replicative Catalytic RNA Systems

Vaidya

2000

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The RNA World hypothesis proposes a period of time during the origins of life in which RNA molecules were the only source of both genotypes and phenotypes. Although a vast amount of evidence has been obtained in support of this hypothesis, a few critical demonstrations are lacking. A most crucial one is a demonstration of self-replication of RNA molecule from prebiotic soup. Previously in the Lehman laboratory, it has been demonstrated that a 198-nucleotide molecule derived from the Azoarcus group I intron can self-assemble from up to four fragments of RNA via recombination. Furthermore, the covalent full-length molecules are catalytically active and can make copies of themselves from the remaining pieces in the solution leading to their autocatalytic growth. I was able to demonstrate how this recombination system can overcome different obstacles and evolve to be an efficient replicating system. I discovered the ability of a single RNA fragment to be multifunctional in a single reaction pathway during RNA recombination events that avoids the necessity of multiple genotypes. I also confirmed the capacity of self-replicating ribozymes to form cooperative catalytic cycles and networks that would potentially prevent informational decay. Finally, I have discovered a recycling phenomenon in the RNA recombination system that exploits dynamic covalent chemistry. Recycling provides the earliest replicating system with adequate concentrations of reagents and ability to explore sequence space. Together these findings have improved our understanding of RNA recombination and bolstered the plausibility of the RNA World.ii DEDICATION To my parents Dil Chandrananda Vaidya and Amina Vaidyaiii ACKNOWLEDGEMENTS

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