Phylogenetic and genetic evidence for base-triples in the catalytic domain of group I introns

Michel, François; Ellington, Andrew D.; Couture, Sandra; Szostak, Jack W.

doi:10.1038/347578a0

Cited by 128 publications

(75 citation statements)

References 14 publications

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“…These mutagenesis data are analogous to those obtained from conventional compensatory mutation analyses. These analyses have most often been used to demonstrate base-pairing interactions within the secondary structure of RNA molecules, but have also been used to establish tertiary interactions (Michel et al 1990;Sampson et al 1990;Murphy and Cech 1994). As a control, double mutants of A210G (which contacts neither residue 269 nor 304 in the crystal structure) in combination with either A269G (A210G<A269G) or A304G (A210G<A304G) did not have any stabilizing effect (Fig.…”

Section: Cooperativity Of A269g and A304g In Enhancing Stabilitymentioning

confidence: 99%

“…U259 has been proposed to interact with the second base pair of the P4 helix in the wild-type Tetrahymena intron (G108 and C213) (Michel et al 1990). In both the Azoarcus and Twort crystal structures, the equivalent residues (G125 in Azoarcus and G117 in Twort) form a major groove contact as predicted (Adams et al 2004;Golden et al 2005).…”

Section: Correlation Of Thermostability Data With Crystal Structuresmentioning

confidence: 99%

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Comparison of crystal structure interactions and thermodynamics for stabilizing mutations in the Tetrahymena ribozyme

Guo¹,

Gooding²,

Cech³

2006

RNA

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Although general mechanisms of RNA folding and catalysis have been elucidated, little is known about how ribozymes achieve structural stability at high temperature. A previous in vitro evolution experiment identified a small number of mutations that significantly increase the thermostability of the tertiary structure of the Tetrahymena ribozyme. Because we also determined the crystal structure of this thermostable ribozyme, we have for the first time the opportunity to compare the structural interactions and thermodynamic contributions of individual nucleotides in a ribozyme. We investigated the contribution of five mutations to thermostability by using temperature gradient gel electrophoresis. Unlike the case with several well-studied proteins, the effects of individual mutations on thermostability of this RNA were highly context dependent. The three most important mutations for thermostability were actually destabilizing in the wild-type background. A269G and A304G contributed to stability only when present as a pair, consistent with their proximity in the ribozyme structure. In an evolutionary context, this work supports and extends the idea that one advantage of protein enzyme systems over an RNA world is the ability of proteins to accumulate stabilizing single-site mutations, whereas RNA may often require much rarer double mutations to improve the stability of both its tertiary and secondary structures.

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Section: Cooperativity Of A269g and A304g In Enhancing Stabilitymentioning

confidence: 99%

Section: Correlation Of Thermostability Data With Crystal Structuresmentioning

confidence: 99%

Comparison of crystal structure interactions and thermodynamics for stabilizing mutations in the Tetrahymena ribozyme

Guo¹,

Gooding²,

Cech³

2006

RNA

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“…If tertiary interactions involving these sites are essential, then some [e.g., triple base pairs (Michel et al 1990)] may be identified with appropriate in vitro selection experiments using variations of the selection method described here. Such experiments are under way in our laboratory.…”

Section: In Vitro Selection Techniques (For Review See Abelsonmentioning

confidence: 99%

In vitro selection of active hairpin ribozymes by sequential RNA-catalyzed cleavage and ligation reactions.

Berzal‐Herranz

Joseph²,

Burke³

1992

Genes Dev.

140

106

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In vitro selection methods provide rapid and extremely powerful tools for elucidating interactions within and between macromolecules. Here, we describe the development of an in vitro selection procedure that permits the rapid isolation and evaluation of functional hairpin ribozymes from a complex pool of sequence variants containing an extremely low frequency of catalytically proficient molecules. We have used this method to analyze the sequence requirements of two regions of the ribozyme-substrate complex: a 7-nucleotide internal loop within the ribozyme that is essential for catalytic function and substrate sequences surrounding the cleavage-ligation site. Results indicate that only 3 of the 16,384 internal loop variants examined have high cleavage and ligation activity and that the ribozyme has a strong requirement for guanosine immediately 3' to the cleavage-ligation site.

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“…For example, the triple-helical scaffold proposed for the P4-P6 region (21) is probably not tested by our experiments. In DNA triplexes, at least two of the three strands can still be cleaved by free radicals produced by chelated Fe(II) (22), so it seems likely that an RNA triplex could remain cleavable by Fe(II)-EDTA if not involved in additional interactions that excluded it from the solvent.…”

mentioning

confidence: 99%

Folding of group I introns from bacteriophage T4 involves internalization of the catalytic core.

Heuer

Chandry

Belfort

et al. 1991

Proc. Natl. Acad. Sci. U.S.A.

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Fe(lH)-EDTA, a solvent-based cleavage reagent that distinguishes between the inside and outside surfaces of a folded RNA molecule, has revealed some of the higherorder folding of the group IB intron from Tetrahymena thermophila pre-rRNA. This reagent has now been used to analyze the bacteriophage T4 sunY and td introns, both of which are members of the group IA subclass. Significant portions of the phylogenetically conserved secondary structure are protected from Fe(ll)-EDTA cleavage. However, the P4 secondary structure element, which is substantially protected in the Tetrahymena intron, is available for cleavage in the two T4 introns. We conclude that a family of catalytic RNAs (ribozymes) that possess similar secondary structures and have similar activities fold into similar but nonidentical tertiary structures that nevertheless serve to internalize portions of the catalytic center. Furthermore, comparison of cleavage patterns of the sunY and td intron RNAs indicates that conserved nucleotides outside as well as within the catalytic core participate in the tertiary structure.Group I introns must fold into a catalytically active structure in order to mediate RNA self-splicing (1, 2). While the secondary structures ofgroup I introns have been established by comparative sequence analysis (3-5), their higher-order folding is only beginning to be understood. Fe(II)-EDTA combines with oxygen to generate free radicals that promote oxidative damage to ribose moieties and result in nucleic acid strand scission (6-9). Because it has little base-sequence specificity and similar reactivity toward single-and doublestranded forms of RNA (10), Fe(II)-EDTA provides a useful probe for the higher-order folding of RNA. In a previous study (6) this methodology was used to analyze the native structure of the L-21 Sca I RNA, a shortened form of the Tetrahymena group IB intron that has enzymatic activity as a sequence-specific endoribonuclease (11). The RNA, in its Mg2+-stabilized active conformation, was found to possess significant cleavage-resistant regions, including portions of elements P3, P4, and P7 that reside in the catalytic center of the ribozyme. Parallel studies with yeast tRNAI'b, a molecule of known structure, supported the interpretation that protection from cleavage was related primarily to low solvent accessibility (6).To assess the generality of the inside-outside folding pattern among group I introns, the bacteriophage T4 sun Y(a split gene of unknown function) and td (thymidylate synthase) mRNA introns were selected for study. The sunY and td introns are self-splicing group IA introns and share common secondary structures as well as considerable primary sequence similarity (ref. 5; Fig. 1). Although the general secondary structure of group IA and IB introns is conserved in the core region consisting of P3, P4, P5, P6, P7, P8, and P9, the sun Y and td intron RNAs differ significantly from the Tetrahymena intron RNA in primary sequence and in the number and arrangement of peripheral secondary structure e...

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Phylogenetic and genetic evidence for base-triples in the catalytic domain of group I introns

Cited by 128 publications

References 14 publications

Comparison of crystal structure interactions and thermodynamics for stabilizing mutations in the Tetrahymena ribozyme

Comparison of crystal structure interactions and thermodynamics for stabilizing mutations in the Tetrahymena ribozyme

In vitro selection of active hairpin ribozymes by sequential RNA-catalyzed cleavage and ligation reactions.

Folding of group I introns from bacteriophage T4 involves internalization of the catalytic core.

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