Group I introns and RNA folding (original) (raw)
Related papers
The ribozyme core of group II introns: a structure in want of partners
Trends in Biochemical Sciences, 2009
Group II introns contain a large ribozyme, which catalyzes self-splicing, and the coding sequence of a reverse transcriptase, the function of which is to cooperate with the ribozyme to achieve genomic mobility. Despite its lack of substrates for both steps of the splicing process, the crystal structure of a group II ribozyme reveals the location of two metal ions most likely to be involved in catalysis; the RNA structure that binds to these ions results from the bending of a local motif by the folding of the rest of the ribozyme. The stage is now set to determine where the intron-encoded protein binds to its partner and whether the spliceosome uses a counterpart of the group II catalytic center to excise nuclear premessenger introns.
Nucleic Acids Research, 2009
Catalytic RNA molecules possess simultaneously a genotype and a phenotype. However, a single RNA genotype has the potential to adopt two or perhaps more distinct phenotypes as a result of differential folding and/or catalytic activity. Such multifunctionality would be particularly significant if the phenotypes were functionally inter-related in a common biochemical pathway. Here, this phenomenon is demonstrated by the ability of the Azoarcus group I ribozyme to function when its canonical internal guide sequence (GUG) has been removed from the 5' end of the molecule, and added back exogenously in trans. The presence of GUG triplets in noncovalent fragments of the ribozyme allow transsplicing to occur in both a reverse splicing assay and a covalent self-assembly assay in which the internal guide sequence (IGS)-less ribozyme can put itself together from two of its component pieces. Analysis of these reactions indicates that a single RNA fragment can perform up to three distinct roles in a reaction: behaving as a portion of a catalyst, behaving as a substrate, and providing an exogenous IGS. This property of RNA to be multifunctional in a single reaction pathway bolsters the probability that a system of self-replicating molecules could have existed in an RNA world during the origins of life on the Earth.
Chem Biol, 1996
Group I introns self-splice via two consecutive trans-esterification reactions in the presence of guanosine cofactor and magnesium ions. Comparative sequence analysis has established that a catalytic core of about 120 nucleotides is conserved in all known group I introns. This core is generally not sufficient for activity, however, and most self-splicing group I introns require nonconserved peripheral elements to stabilize the complete three-dimensional (3D) structure. The physico-chemical properties of group I introns make them excellent systems for unraveling the structural basis of the RNA-RNA interactions responsible for promoting the self-assembly of complex RNAs.
European Journal of Biochemistry, 2004
DiGIR2 is the group I splicing-ribozyme of the mobile twinribozyme intron Dir.S956-1, present in Didymium nuclear ribosomal DNA. DiGIR2 is responsible for intron excision, exon ligation, 3¢-splice site hydrolysis, and full-length intron RNA circle formation. We recently reported that DiGIR2 splicing (intron excision and exon ligation) competes with hydrolysis and subsequent full-length intron circularization. Here we present experimental evidence that hydrolysis at the 3¢-splice site in DiGIR2 is dependent on structural elements within the P9 subdomain not involved in splicing. Whereas the GCGA tetra-loop in P9b was found to be important in hydrolytic cleavage, probably due to tertiary RNA-RNA interactions, the P9.2 hairpin structure was found to be essential for hydrolysis. The most important positions in P9.2 include three adenosines in the terminal loop (L9.2) and a consensus kink-turn motif in the proximal stem. We suggest that the L9.2 adenosines and the kink-motif represent key regulatory elements in the splicing and hydrolytic reaction pathways.
Productive folding to the native state by a group II intron ribozyme
Journal of Molecular Biology, 2002
Group II introns are large catalytic RNA molecules that fold into compact structures essential for the catalysis of splicing and intron mobility reactions. Despite a growing body of information on the folded state of group II introns at equilibrium, there is currently no information on the folding pathway and little information on the ionic requirements for folding. Folding isotherms were determined by hydroxyl radical footprinting for the 32 individual protections that are distributed throughout a group II intron ribozyme derived from intron ai5g. The isotherms span a similar range of Mg 2 concentrations and share a similar index of cooperativity. Time-resolved hydroxyl radical footprinting studies show that all regions of the ribozyme fold slowly and with remarkable synchrony into a single catalytically active structure at a rate comparable to those of other ribozymes studied thus far. The rate constants for the formation of tertiary contacts and recovery of catalytic activity are identical within experimental error. Catalytic activity analyses in the presence of urea provide no evidence that the slow folding of the ai5g intron is attributable to the presence of unproductive kinetic traps along the folding pathway. Taken together, the data suggest that the rate-limiting step for folding of group II intron ai5g occurs early along the reaction pathway. We propose that this behavior resembles protein folding that is limited in rate by high contact order, or the need to form key tertiary interactions from partners that are located far apart in the primary or secondary structure.
RNA Folding Causes Secondary Structure Rearrangement
Proceedings of The National Academy of Sciences, 1998
The secondary structure of the P5abc subdomain (a 56-nt RNA) of the Tetrahymena thermophila group I intron ribozyme has been determined by NMR. Its base pairing in aqueous solution in the absence of magnesium ions is significantly different from the RNA in a crystal but is consistent with thermodynamic predictions. On addition of magnesium ions, the RNA folds into a tertiary structure with greatly changed base pairing consistent with the crystal structure: three Watson-Crick base pairs, three G⅐U base pairs, and an extra-stable tetraloop are lost. The common assumption that RNA folds by first forming secondary structure and then forming tertiary interactions from the unpaired bases is not always correct.
Solution structure of domain 5 of a group II intron ribozyme reveals a new RNA motif
Nature Structural & Molecular Biology, 2004
Domain 5 (D5) is the central core of group II intron ribozymes. Many base and backbone substituents of this highly conserved hairpin participate in catalysis and are crucial for binding to other intron domains. We report the solution structures of the 34-nucleotide D5 hairpin from the group II intron ai5 gamma in the absence and presence of divalent metal ions. The bulge region of D5 adopts a novel fold, where G26 adopts a syn conformation and flips down into the major groove of helix 1, close to the major groove face of the catalytic AGC triad. The backbone near G26 is kinked, exposing the base plane of the adjacent A-U pair to the solvent and causing bases of the bulge to stack intercalatively. Metal ion titrations reveal strong Mg(2+) binding to a minor groove shelf in the D5 bulge. Another distinct metal ion-binding site is observed along the minor groove side of the catalytic triad, in a manner consistent with metal ion binding in the ribozyme active site.
The Right Angle (RA) Motif: A Prevalent Ribosomal RNA Structural Pattern Found in Group I Introns
Journal of Molecular Biology, 2012
The right angle (RA) motif, previously identified in the ribosome and used as a structural module for nano-construction, is a recurrent structural motif of 13 nucleotides that establishes a 90° bend between two adjacent helices. Comparative sequence analysis was used to explore the sequence space of the RA motif within ribosomal RNAs in order to define its canonical sequence space signature. We investigated the sequence constraints associated with the RA signature using several artificial self-assembly systems. Thermodynamic and topological investigations of sequence variants associated with the RA motif in both minimal and expanded structural contexts reveal that the presence of a helix at the 3′ end of the RA motif increases the thermodynamic stability and rigidity of the resulting 3-helix junction domain. A search for the RA in naturally occurring RNAs as well as its experimental characterization led to the identification of the RA in groups IC1 and ID intron ribozymes, where it is suggested to play an integral role in stabilizing peripheral structural domains. The present study exemplifies the need of empirical analysis of RNA structural motifs for facilitating the rational design and structure prediction of RNAs.
Now on display: a gallery of group II intron structures at different stages of catalysis
Mobile DNA, 2013
Group II introns are mobile genetic elements that self-splice and retrotranspose into DNA and RNA. They are considered evolutionary ancestors of the spliceosome, the ribonucleoprotein complex essential for pre-mRNA processing in higher eukaryotes. Over a 20-year period, group II introns have been characterized first genetically, then biochemically, and finally by means of X-ray crystallography. To date, 17 crystal structures of a group II intron are available, representing five different stages of the splicing cycle. This review provides a framework for classifying and understanding these new structures in the context of the splicing cycle. Structural and functional implications for the spliceosome are also discussed.
Influence of substrate structure on in vitro ribozyme activity of a group II intron
Rna-a Publication of The Rna Society, 1998
Substrate sequences surrounding catalytic RNAs but not involved in specific, conserved interactions can severely interfere with in vitro ribozyme activity. Using model group II intron precursor transcripts with truncated or randomized exon sequences, we show that unspecific sequences within the 59 exon are particularly prone to inhibit both the forward and the reverse first splicing step (branching). Using in vitro selection, we selected efficient 59 exons for the reverse branching reaction. Precursor RNAs carrying these selected 59 exons reacted more homogeneously and faster than usual model precursor transcripts. This suggests that unfavorable structures induced by the 59 exon can introduce a folding step that limits the rate of in vitro self-splicing. These results stress how critical is the choice of the sequences retained or discarded when isolating folding domains from their natural sequence environments. Moreover, they suggest that exon sequences not involved in specific interactions are more evolutionarily constrained with respect to splicing than previously envisioned.