New loop-loop tertiary interactions in self-splicing introns of subgroup IC and ID: a complete 3D model of the Tetrahymena thermophila ribozyme (original) (raw)
Related papers
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.
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.
Biochemistry, 1996
Structural studies were performed on synthetic oligonucleotides with sequences corresponding to the P4/P6 and J3/4, J6/7 regions of the self-splicing group I intron of the bacteriophage T4 nrdB pre-mRNA, which correspond to the proposed triple-helical domain in the Tetrahymena thermophila intron. A 23-mer RNA was synthesized as a mixed ribo-deoxyribo oligonucleotide, modeling an expected basepaired region P4 along with the J3/4 and P6 (5′-end bases of P6) regions. A third strand modeling the 3′-end bases of P6 and J6/7 regions, with which a triple helix may form, was synthesized as a pure oligoribonucleotide (7-mer RNA). The interactions of these oligonucleotides have been characterized by UV and circular dichroism (CD) spectroscopy. The results show that the 23-mer RNA forms a stable hairpin modeling the P4 base-paired region. Triple helix association between the 23-mer RNA hairpin and the 7-mer RNA single strand was detected by CD in the presence of Mg 2+ (>5 mM) but not in presence of a monovalent cation like Na + (up to 500 mM). Studies on selected variants of both 7-mer and 23-mer RNAs were carried out. The results show that for the association of the two partner strands not only the formation of P6 helix but also triplet interactions between the two strands are required. The association of the two strands in general follow a pattern predicted by comparative sequence analysis. Parallel studies on pure oligodeoxyribonucleotides having base sequences corresponding to those of the oligoribonucleotides showed no evidence of association under similar conditions, which could indicate that the 2′-hydroxyl groups of the riboses might play an important role in hydrogen bonding to form the required nucleoside triples. Molecular modeling studies on the proposed "plaited triple helix" formed by the association of the 23-mer RNA hairpin and 7-mer RNA single strand showed that the structure is sterically and energetically feasible.
Molecular cell, 2008
Group II introns are self-splicing ribozymes believed to be the ancestors of spliceosomal introns. Many group II introns encode reverse transcriptases that promote both RNA splicing and intron mobility to new genomic sites. Here we used a circular permutation and cross-linking method to establish sixteen intramolecular distance relationships within the mobile Lactococcus lactis Ll.LtrB-ΔORF intron. Using these new constraints together with thirteen established tertiary interactions and eight published cross-links, we modeled a complete three-dimensional structure of the intron. We also used the circular permutation strategy to map RNA-protein interaction sites through fluorescence quenching and cross-linking assays. Our model provides a comprehensive structural framework for understanding the function of group II ribozymes, their natural structural variations, and the mechanisms by which the intron-encoded protein promotes RNA splicing and intron mobility. The model also suggests an arrangement of active site elements that may be conserved in the spliceosome.
Journal of Molecular Biology, 1995
The mitochondrial genes of the yeast Saccharomyces cerevisiae are often Laboratoire du Génétique Moléculaire, Ecole Normale interrupted by introns defined as either group I or group II. Some of the Supérieure, 46 rue d'Ulm introns contained within the precursor RNAs of these genes will self splice 75230 Paris, France in vitro. The fourth introns of apocytochrome b (bi4) and cytochrome oxidase (ai4) are group I introns that do not self splice in vitro, even though they can fold into the same RNA secondary structures that are characteristic of the self-splicing introns. They require an intron-encoded maturase protein and a nuclear-encoded protein (a tRNA Leu synthetase) for splicing in vivo. We have divided these introns into several sequence or structural elements and assayed them individually for their ability to support self-splicing activity. This was done by replacing the equivalent elements from the self-splicing intron from Tetrahymena thermophila with the mitochondrial elements. These intron chimeras show that peripheral sequences and the elements that define the splice sites are adequate for self-splicing activity but that the central portions containing the catalytic cores of ai4 and bi4 are deficient; these cores are the likely targets of the splicing proteins. In addition, the catalytic activity of the Tetrahymena intron is remarkably resistant to the structural alterations that we have introduced; this suggests that this technique will be of general utility for studying the structural and functional relationships of elements contained within different 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.
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.
Group I introns and RNA folding
Biochemical Society Transactions, 2002
Before the discovery of catalytic RNA, tRNA molecules were the most studied RNA molecules for understanding RNA folding. Afterwards, group I introns, because of their stability and the fact that structural folding could be monitored by following their catalytic activity, became the molecule of choice for studying RNA architecture and folding. A major advantage of group I introns for studying the catalytic activity of RNA molecules is that catalytic activity is triggered by the addition of external guanosine co-factors. The self-splicing activity can therefore be precisely controlled. Using group I introns, several RNA motifs central to RNA-RNA self-assembly and folding were discovered. The analysis of the recent X-ray structures of the rRNA subunits indicates that several motifs present in the ribosome occur also in various group I introns.
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.
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.