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Group II introns are self-splicing, mobile genetic elements that have fundamentally influenced the organization of terrestrial genomes. These large ribozymes remain important for gene expression in almost all forms of bacteria and eukaryotes and they are believed to share a common ancestry with the eukaryotic spliceosome that is required for processing all nuclear pre-mRNAs. The three-dimensional structure of a group IIC intron was recently determined by X-ray crystallography, making it possible to visualize the active site and the elaborate network of tertiary interactions that stabilize the molecule. Here we describe the molecular features of the active site in detail and evaluate their correspondence with prior biochemical, genetic, and phylogenetic analyses on group II introns. In addition, we evaluate the structural significance of RNA motifs within the intron core, such as the major-groove triple helix and the domain 5 bulge. Having combined what is known about the group II intron core, we then compare it with known structural features of U6 snRNA in the eukaryotic spliceosome. This analysis leads to a set of predictions for the molecular structure of the spliceosomal active site. 相似文献
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The X-ray crystal structure of an excised group II self-splicing intron was recently solved by the Pyle group. Here we review some of the notable features of this structure and what they may tell us about the catalytic active site of the group II ribozyme and potentially the spliceosome. The new structure validates the central role of domain V in both the structure and catalytic function of the ribozyme and resolves several outstanding puzzles raised by previous biochemical, genetic and structural studies. While lacking both exons as well as the cleavage sites and nucleophiles, the structure reveals how a network of tertiary interactions can position two divalent metal ions in a configuration that is ideal for catalysis. 相似文献
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Metal ions promote both RNA folding and catalysis, thus being essential in stabilizing the structure and determining the function of large RNA molecules, including group II introns. The latter are self-splicing metalloribozymes, containing a heteronuclear four-metal-ion center within the active site. In addition to these catalytic ions, group II introns bind many other structural ions, including delocalized ions that bind the RNA diffusively and well-ordered ions that bind the RNA tightly with high occupancy. The latter ions, which can be studied by biophysical methods, have not yet been analyzed systematically. Here, we compare crystal structures of the group IIC intron from Oceanobacillus iheyensis and classify numerous site-bound ions, which are primarily localized in the intron core and near long-range tertiary contacts. Certain ion-binding sites resemble motifs observed in known RNA structures, while others are idiosyncratic to the group II intron. Particularly interesting are (1) ions proximal to the active site, which may participate in splicing together with the catalytic four-metal-ion center, (2) organic ions that bind regions predicted to interact with intron-encoded proteins, and (3) unusual monovalent ions bound to GU wobble pairs, GA mismatches, the S-turn, the tetraloop-receptor, and the T-loop. Our analysis extends the general principles by which ions participate in RNA structural organization and it will aid in the determination and interpretation of future RNA structures. 相似文献
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Although the active site of group I introns is phylogenetically conserved, subclasses of introns have evolved different mechanisms of stabilizing the catalytic core. Large introns contain weakly conserved 'peripheral' domains that buttress the core through predicted interhelical contacts, while smaller introns use loop-helix interactions for stability. In all cases, specific and non-specific magnesium ion binding accompanies folding into the active structure. Whether similar RNA-RNA and RNA-magnesium ion contacts play related functional roles in different introns is not clear, particularly since it can be difficult to distinguish interactions directly involved in catalysis from those important for RNA folding. Using phosphorothioate interference with RNA activity and structure in the small (249 nt) group I intron from Anabaena, we used two independent assays to detect backbone phosphates important for catalysis and those involved in intron folding. Comparison of the interference sites identified in each assay shows that positions affecting catalysis cluster primarily in the conserved core of the intron, consistent with conservation of functionally important phosphates, many of which are magnesium ion binding sites, in diverse group I introns, including those from Azoarcus and Tetrahymena. However, unique sites of folding interference located outside the catalytic core imply that different group I introns, even within the same subclass, use distinct sets of tertiary interactions to stabilize the structure of the catalytic core. 相似文献
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CRS1, a chloroplast group II intron splicing factor, promotes intron folding through specific interactions with two intron domains 总被引:4,自引:0,他引:4
Group II introns are ribozymes that catalyze a splicing reaction with the same chemical steps as spliceosome-mediated splicing. Many group II introns have lost the capacity to self-splice while acquiring compensatory interactions with host-derived protein cofactors. Degenerate group II introns are particularly abundant in the organellar genomes of plants, where their requirement for nuclear-encoded splicing factors provides a means for the integration of nuclear and organellar functions. We present a biochemical analysis of the interactions between a nuclear-encoded group II splicing factor and its chloroplast intron target. The maize (Zea mays) protein Chloroplast RNA Splicing 1 (CRS1) is required specifically for the splicing of the group II intron in the chloroplast atpF gene and belongs to a plant-specific protein family defined by a recently recognized RNA binding domain, the CRM domain. We show that CRS1's specificity for the atpF intron in vivo can be explained by CRS1's intrinsic RNA binding properties. CRS1 binds in vitro with high affinity and specificity to atpF intron RNA and does so through the recognition of elements in intron domains I and IV. These binding sites are not conserved in other group II introns, accounting for CRS1's intron specificity. In the absence of CRS1, the atpF intron has little uniform tertiary structure even at elevated [Mg2+]. CRS1 binding reorganizes the RNA, such that intron elements expected to be at the catalytic core become less accessible to solvent. We conclude that CRS1 promotes the folding of its group II intron target through tight and specific interactions with two peripheral intron segments. 相似文献
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Despite the biological importance of self-splicing group II introns, little is known about their structural organization. Synthetic incorporation of site-specific photo-cross-linkers within catalytic domains resulted in functional distance constraints that, when combined with known tertiary interactions, provide a three-dimensional view of the active intron architecture. All functionalities important for both steps of splicing are proximal before the first step, suggestive of a single active-site region for group II intron catalysis. 相似文献
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Group I introns are catalytic RNAs capable of orchestrating two sequential phosphotransesterification reactions that result in self-splicing. To understand how the group I intron active site facilitates catalysis, we have solved the structure of an active ribozyme derived from the orf142-I2 intron from phage Twort bound to a four-nucleotide product RNA at a resolution of 3.6 A. In addition to the three conserved domains characteristic of all group I introns, the Twort ribozyme has peripheral insertions characteristic of phage introns. These elements form a ring that completely envelops the active site, where a snug pocket for guanosine is formed by a series of stacked base triples. The structure of the active site reveals three potential binding sites for catalytic metals, and invokes a role for the 2' hydroxyl of the guanosine substrate in organization of the active site for catalysis. 相似文献
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Jennifer F Swisher Linhui J Su Michael Brenowitz Vernon E Anderson Anna Marie Pyle 《Journal of molecular biology》2002,315(3):297-310
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 ai5gamma. 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 ai5gamma 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 ai5gamma 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. 相似文献
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Group II intron splicing in chloroplasts: identificationof mutations determining intron stability and fate of exon RNA.
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In order to investigate in vivo splicing of group II introns in chloroplasts, we previously have integrated the mitochondrial intron rI1 from the green alga Scenedesmus obliquus into the Chlamydomonas chloroplast tscA gene. This construct allows a functional analysis of conserved intron sequences in vivo, since intron rI1 is correctly spliced in chloroplasts. Using site-directed mutagenesis, deletions of the conserved intron domains V and VI were performed. In another set of experiments, each possible substitution of the strictly conserved first intron nucleotide G1 was generated, as well as each possible single and double mutation of the tertiary base pairing gamma-gamma ' involved in the formation of the intron's tertiary RNA structure. In most cases, the intron mutations showed the same effect on in vivo intron splicing efficiency as they did on the in vitro self-splicing reaction, since catalytic activity is provided by the intron RNA itself. In vivo, all mutations have additional effects on the chimeric tscA -rI1 RNA, most probably due to the role played by trans -acting factors in intron processing. Substitutions of the gamma-gamma ' base pair lead to an accumulation of excised intron RNA, since intron stability is increased. In sharp contrast to autocatalytic splicing, all point mutations result in a complete loss of exon RNA, although the spliced intron accumulates to high levels. Intron degradation and exon ligation only occur in double mutants with restored base pairing between the gamma and gamma' sites. Therefore, we conclude that intron degradation, as well as the ligation of exon-exon molecules, depends on the tertiary intron structure. Furthermore, our data suggest that intron excision proceeds in vivo independent of ligation of exon-exon molecules. 相似文献
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Group II introns are large catalytic RNA molecules that act as mobile genetic elements. They were initially identified in the organelle genomes of lower eukaryotes and plants, and it has been suggested that they are the progenitors of nuclear spliceosomal introns. Group II self-splicing introns were shown to be present in bacteria in 1993, since when the various bacterial genome sequencing projects have led to a significant increase in the number of group II intron sequences present in databases. However, few of these introns have been characterized, and most were identified on the basis of their intron-encoded protein (IEP), with little data available concerning their ribozyme/RNA structure. Their frequency in prokaryotes is also unknown. We attempt here to provide a first comprehensive review of bacterial group II introns based on recent genome sequencing data and mechanistic studies. 相似文献
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Lydia M. Contreras Tao Huang Carol Lyn Piazza Dorie Smith Guosheng Qu Grant Gelderman Jeffrey P. Potratz Rick Russell Marlene Belfort 《RNA (New York, N.Y.)》2013,19(11):1497-1509
The influence of the cellular environment on the structures and properties of catalytic RNAs is not well understood, despite great interest in ribozyme function. Here we report on ribosome association of group II introns, which are ribozymes that are important because of their putative ancestry to spliceosomal introns and retrotransposons, their retromobility via an RNA intermediate, and their application as gene delivery agents. We show that group II intron RNA, in complex with the intron-encoded protein from the native Lactoccocus lactis host, associates strongly with ribosomes in vivo. Ribosomes have little effect on intron ribozyme activities; rather, the association with host ribosomes protects the intron RNA against degradation by RNase E, an enzyme previously shown to be a silencer of retromobility in Escherichia coli. The ribosome interacts strongly with the intron, exerting protective effects in vivo and in vitro, as demonstrated by genetic and biochemical experiments. These results are consistent with the ribosome influencing the integrity of catalytic RNAs in bacteria in the face of degradative nucleases that regulate intron mobility. 相似文献
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A CRM domain protein functions dually in group I and group II intron splicing in land plant chloroplasts 总被引:3,自引:0,他引:3
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The CRM domain is a recently recognized RNA binding domain found in three group II intron splicing factors in chloroplasts, in a bacterial protein that associates with ribosome precursors, and in a family of uncharacterized proteins in plants. To elucidate the functional repertoire of proteins with CRM domains, we studied CFM2 (for CRM Family Member 2), which harbors four CRM domains. RNA coimmunoprecipitation assays showed that CFM2 in maize (Zea mays) chloroplasts is associated with the group I intron in pre-trnL-UAA and group II introns in the ndhA and ycf3 pre-mRNAs. T-DNA insertions in the Arabidopsis thaliana ortholog condition a defective-seed phenotype (strong allele) or chlorophyll-deficient seedlings with impaired splicing of the trnL group I intron and the ndhA, ycf3-int1, and clpP-int2 group II introns (weak alleles). CFM2 and two previously described CRM proteins are bound simultaneously to the ndhA and ycf3-int1 introns and act in a nonredundant fashion to promote their splicing. With these findings, CRM domain proteins are implicated in the activities of three classes of catalytic RNA: group I introns, group II introns, and 23S rRNA. 相似文献
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Group II introns are self-splicing RNA molecules that also behave as mobile genetic elements. The secondary structure of group II intron RNAs is typically described as a series of six domains that project from a central wheel. Most structural and mechanistic analyses of the intron have focused on domains 1 and 5, which contain the residues essential for catalysis, and on domain 6, which contains the branch-point adenosine. Domains 2 and 3 (D2, D3) have been shown to make important contributions to intronic activity; however, information about their function is quite limited. To elucidate the role of D2 and D3 in group II ribozyme catalysis, we built a series of multi-piece ribozyme constructs based on the ai5gamma group II intron. These constructs are designed to shed light on the roles of D2 and D3 in some of the major reactions catalyzed by the intron: 5'-exon cleavage, branching, and substrate hydrolysis. Reactions with these constructs demonstrate that D3 stimulates the chemical rate constant of group II intron reactions, and that it behaves as a form of catalytic effector. However, D3 is unable to associate independently with the ribozyme core. Docking of D3 is mediated by a short duplex that is found at the base of D2. In addition to recruiting D3 into the core, the D2 stem directs the folding of the adjacent j(2/3) linker, which is among the most conserved elements in the group II intron active site. In turn, the D2 stem contributes to 5'-splice site docking and ribozyme conformational change. Nucleotide analog interference mapping suggests an interaction between the D2 stem and D3 that builds on the known theta-theta' interaction and extends it into D3. These results establish that D3 and the base of D2 are key elements of the group II intron core and they suggest a hierarchy for active-site assembly. 相似文献
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Metal ions play key roles in the folding and function for many structured RNAs, including group I introns. We determined the X-ray crystal structure of the Azoarcus bacterial group I intron in complex with its 5' and 3' exons. In addition to 222 nucleotides of RNA, the model includes 18 Mg(2+) and K(+) ions. Five of the metals bind within 12 A of the scissile phosphate and coordinate the majority of the oxygen atoms biochemically implicated in conserved metal-RNA interactions. The metals are buried deep within the structure and form a multiple metal ion core that is critical to group I intron structure and function. Eight metal ions bind in other conserved regions of the intron structure, and the remaining five interact with peripheral structural elements. Each of the 18 metals mediates tertiary interactions, facilitates local bends in the sugar-phosphate backbone or binds in the major groove of helices. The group I intron has a rich history of biochemical efforts aimed to identify RNA-metal ion interactions. The structural data are correlated to the biochemical results to further understand the role of metal ions in group I intron structure and function. 相似文献
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The 3' regions of several group II introns within the mitochondrial genes nad1 and nad7 show unexpected sequence divergence among flowering plants, and the core domains 5 and 6 are predicted to have weaker helical structure than those in self-splicing group II introns. To assess whether RNA editing improves helical stability by the conversion of A-C mispairs to A-U pairs, we sequenced RT-PCR amplification products derived from excised intron RNAs or partially spliced precursors. Only in some cases was editing observed to strengthen the predicted helices. Moreover, the editing status within nad1 intron 1 and nad7 intron 4 was seen to differ among plant species, so that homologous intron sequences shared lower similarity at the RNA level than at the DNA level. Plant-specific variation was also seen in the length of the linker joining domains 5 and 6 of nad7 intron 3; it ranged from 4 nt in wheat to 11 nt in soybean, in contrast to the 2-4 nt length typical of classical group II introns. However, this intron is excised as a lariat structure with a domain 6 branchpoint adenosine. Our observations suggest that the core structures and sequences of these plant mitochondrial introns are subject to less stringent evolutionary constraints than conventional group II introns. 相似文献