SHAPE analysis of long-range interactions reveals extensive and thermodynamically preferred misfolding in a fragile group I intron RNA

Most functional RNAs require proteins to facilitate formation of their active structures. In the case of the yeast bI3 group I intron, splicing requires binding by two proteins, the intron-encoded bI3 maturase and the nuclear encoded Mrs1. Here, we use selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE) chemistry coupled with analysis of point mutants to map long-range interactions in this RNA. This analysis reveals two critical features of the free RNA state. First, the catalytic intron is separated from the flanking exons via a stable anchoring helix. This anchoring helix creates an autonomous structural domain for the intron and functions to prevent misfolding with the flanking exons. Second, the thermodynamically most stable structure for the free RNA is not consistent with the catalytically active conformation as phylogenetically conserved elements form stable, non-native structures. These results highlight a fragile bI3 RNA for which binding by protein cofactors functions to promote extensive secondary structure rearrangements that are an obligatory prerequisite for forming the catalytically active tertiary structure.     

Chemical interrogation of mismatches in DNA-DNA and DNA-RNA duplexes under nonstringent conditions by selective 2'-amine acylation

2'-Amine-substituted nucleotides in hybridized duplexes can be chemically tagged in an acylation reaction that is faster for mismatched or flexible nucleotides than for residues constrained by base pairing. Here we explore mismatch and hybridization detection using probe oligodeoxynucleotides containing single 2'-aminocytidine or -uridine nucleotides annealed to DNA or RNA targets under nonstringent conditions, below T(m). Consistent with a mechanism in which 2'-amine acylation is gated by local nucleotide flexibility, we find that efficient acylation is correlated with formation of weaker or fewer hydrogen bonds in base pair mismatches. Using 2'-aminocytidine-containing probes annealed to both DNA and RNA targets, mismatches are reliably detected as rapid selective acylation of the 2'-amine group in two sequence contexts. For probe oligonucleotides containing 2'-aminouridine residues, good discrimination between U-A base pairs and U-G mismatches could be obtained for DNA-DNA but not for DNA-RNA duplexes upon the introduction of a single 2'-O-Me group 5' to the 2'-amino nucleotide. The 2'-O-Me group introduces a structural perturbation, presumably to a more A-form-like structure, that exaggerates local flexibility at mismatches in DNA strands. Thus, 2'-amine acylation can be used to interrogate all possible mismatches in DNA-DNA duplexes and mismatches involving 2'-amine-substituted cytidine nucleotides in DNA-RNA heteroduplexes. Applications of this chemistry include detecting and chemically proofreading single nucleotide polymorphisms in both DNA and RNA targets and quantifying absolute amounts of RNA.     

Determination of functional domains in intron bI1 of yeast mitochondrial RNA by studies of mitochondrial mutations and a nuclear suppressor.

The sequence of intron 1 in the cob gene in mtDNA (bI1) of the yeast strain 777-3A has been determined. Furthermore, we have performed a systematic search for complementary sequence stretches within this intron RNA, and within the RNA of intron 5 gamma of the oxi3 gene (aI5 gamma) which shares distinctive sequences with bI1. Possible secondary structure models derived from this analysis show nearly identical core structures for bI1 and aI5 gamma RNA with conserved sequence stretches in prominent positions. These core structures are similar to those previously reported for RNAs of introns having very limited sequence homology with bI1 and aI5 gamma. In two mutants which are defective in bI1 excision from cob pre-mRNA, nucleotide sequence alterations in bI1 have been determined. One mutation (G5049) apparently affects the stability of a hybrid stretch in the proposed secondary structure of bI1 RNA whereas the other one (M1301), a deletion of one A in a run of five As, affects a sequence which is conserved in bI1 and aI5 gamma and is involved in the formation of a distinct secondary structure. Out of seven revertants of M1301, three were found to have restored the wild-type bI1 sequence AAAAA, three others had the related sequence AAAAG which is functionally indistinguishable from wild-type, whereas one revertant had a nuclear mutation which suppresses the splicing defect exerted by the mitochondrial mutation M1301. This nuclear suppressor (SUP-101) is allele specific and dominant. The possible role of the sequence affected by M1301 in terms of a recognition site for a nuclear gene product will be discussed.     

Mechanics of DNA flexibility visualized by selective 2'-amine acylation at nucleotide bulges

We used selective acylation of 2'-amine-substituted nucleotides to visualize local backbone conformations that occur preferentially at bulged sites in DNA duplexes. 2'-Amine acylation reports local nucleotide flexibility because unconstrained 2'-amino nucleotides more readily reach a reactive conformation in which the amide-forming transition state is stabilized by interactions between the amine nucleophile and the adjacent 3'-phosphodiester group. Bulged 2'-amine-substituted cytidine nucleotides react approximately 20-fold more rapidly than nucleotides constrained by base-pairing at 35 degrees C. In contrast, base-paired 2'-amine-substituted nucleotides flanked by a 5' or 3' bulge react two- or six-fold more rapidly, respectively, than the perfectly paired duplex. The relative lack of 2'-amine reactivity for nucleotides adjacent to a DNA bulge emphasizes, first, that structural perturbations do not extend significantly into the flanking duplex structure. Second, the exquisite sensitivity towards very local perturbations in nucleic acid structure suggests that 2'-amine acylation can be used to chemically interrogate deletion mutations in DNA. Finally, these data support the mechanical interpretation that the reactive ribose conformation for 2'-amine acylation requires that the base lies out of the helix and in the major groove, a mechanistic insight useful for designing 2'-amine-based sensors.     

CBP2 protein promotes in vitro excision of a yeast mitochondrial group I intron.

The terminal intron (bI2) of the yeast mitochondrial cytochrome b gene is a group I intron capable of self-splicing in vitro at high concentrations of Mg2+. Excision of bI2 in vivo, however, requires a protein encoded by the nuclear gene CBP2. The CBP2 protein has been partially purified from wild-type yeast mitochondria and shown to promote splicing at physiological concentrations of Mg2+. The self-splicing and protein-dependent splicing reactions utilized a guanosine nucleoside cofactor, the hallmark of group I intron self-splicing reactions. Furthermore, mutations that abolished the autocatalytic activity of bI2 also blocked protein-dependent splicing. These results indicated that protein-dependent excision of bI2 is an RNA-catalyzed process involving the same two-step transesterification mechanism responsible for self-splicing of group I introns. We propose that the CBP2 protein binds to the bI2 precursor, thereby stabilizing the catalytically active structure of the RNA. The same or a similar RNA structure is probably induced by high concentrations of Mg2+ in the absence of protein. Binding of the CBP2 protein to the unspliced precursor was supported by the observation that the protein-dependent reaction was saturable by the wild-type precursor. Protein-dependent splicing was competitively inhibited by excised bI2 and by a splicing-defective precursor with a mutation in the 5' exon near the splice site but not by a splicing-defective precursor with a mutation in the core structure. Binding of the CBP2 protein to the precursor RNA had an effect on the 5' splice site helix, as evidenced by suppression of the interaction of an exogenous dinucleotide with the internal guide sequence.     

Leucyl-tRNA Synthetase-dependent and -independent Activation of a Group I Intron

Leucyl-tRNA synthetase (LeuRS) is an essential RNA splicing factor for yeast mitochondrial introns. Intracellular experiments have suggested that it works in collaboration with a maturase that is encoded within the bI4 intron. RNA deletion mutants of the large bI4 intron were constructed to identify a competently folded intron for biochemical analysis. The minimized bI4 intron was active in RNA splicing and contrasts with previous proposals that the canonical core of the bI4 intron is deficient for catalysis. The activity of the minimized bI4 intron was enhanced in vitro by the presence of the bI4 maturase or LeuRS.Although the aminoacyl-tRNA synthetases (aaRSs)⁶ are best known for their role in protein synthesis, many have functionally expanded and are essential to a wide range of other cellular activities that are unrelated to tRNA aminoacylation (1). The class I aaRSs, leucyl- (LeuRS or NAM2) and tyrosyl-tRNA synthetase (TyrRS or CYT-18) are required for RNA splicing of cognate group I introns in the mitochondria of certain lower eukaryotes (2). In yeast, processing of two related group I introns called bI4 and aI4α (Fig. 1) from the cob and cox1α genes, respectively, require yeast mitochondrial LeuRS (3, 4). Likewise, expression of Neurospora crassa mitochondrial genes, such as those for the large ribosomal RNA, is dependent on TyrRS for excising group I introns (5).Open in a separate windowFIGURE 1.Predicted secondary structures of the bI4 and aI4α group I introns. The secondary structure of the canonical core was based on previous predictions (19). Solid bold lines indicate linear connectivities of the nucleic acid strand with arrowheads oriented in the 5′ to 3′ direction. The dashed lines represent putative tertiary interactions. Dotted lines with numbers identify insertions where secondary structures were ambiguous. Arrows in the P1 and P9 domain show splice sites, whereas boxed nucleotides are paired regions.LeuRS facilitates RNA splicing in concert with a bI4 maturase that is encoded within the bI4 intron. Genetic investigations showed that an inactivated bI4 maturase resulting in deficient splicing activity of the bI4 and aI4α group I introns can be rescued by a suppressor mutation of LeuRS to restore mitochondrial respiration (4, 6). In addition, the splicing defect can be compensated by a mutant aI4α DNA endonuclease that is closely related to the bI4 maturase (7, 8).Previously, we used intracellular three-hybrid assays to demonstrate that LeuRS and bI4 maturase can independently bind to the bI4 intron and stimulate RNA splicing activity in the non-physiological yeast nucleus compartment (9). RNA-dependent two-hybrid assays also supported that the bI4 intron could simultaneously bind both the bI4 maturase and LeuRS. In this case, the RNA was co-expressed with LeuRS and bI4 maturase that was fused to either LexA or B42 to generate a two-hybrid response. This suggested that the bI4 intron was bridging these two protein splicing factors. In either the RNA-dependent two-hybrid or three-hybrid assays, bI4 intron splicing occurred only in the presence of LeuRS or bI4 maturase or both.We hypothesized that the bI4 maturase and LeuRS bind to distinct sites of the bI4 intron to form a ternary complex and promote efficient splicing activity. However, the functional basis of the collaboration between these two splicing cofactors or how either of them promotes RNA splicing remains unclear.We sought to characterize the respective splicing roles of the bI4 maturase and LeuRS via biochemical investigations. Previous attempts to develop an in vitro splicing assay for the bI4 intron or its closely related aI4α intron have failed (10, 11). It was hypothesized that the long length of the bI4 intron (∼1600 nucleotides) and its highly A:U-rich content (∼80%) hindered RNA folding in vitro as well as stabilization of its competent structure.Efforts to produce an active form of the bI4 intron have relied on building chimeric group I introns by interchanging RNA domains with the more stable Tetrahymena thermophila group I intron (11). Based on these results, it was proposed that the catalytic core of the bI4 group I intron was inherently defective (11). In this case, the group I intron would be expected to be completely dependent on its protein splicing factors similar to the bI3 intron that relies on the bI3 maturase and Mrs1 for activity (12). Thus, it was hypothesized that the bI4 maturase and/or LeuRS splicing factors aided the bI4 group I intron by targeting its core region to compensate for these deficiencies.We focused our efforts on re-designing the bI4 intron to develop a minimized molecule that might be competent for splicing. Because both the bI4 and aI4α group I introns rely on the bI4 maturase and LeuRS for their splicing activity, we compared their secondary structures to identify and eliminate peripheral regions outside of their catalytic cores. A small active derivative of the bI4 intron, comprised of just 380 nucleotides primarily from the canonical core, was generated. Thus, we show that, in and of itself, the canonical core of this group I intron is competent for splicing. Both the bI4 maturase and LeuRS enhance the splicing activity of the minimized bI4 intron. However, it is possible that protein-dependent splicing of the bI4 intron represents an intermediate evolutionary step in which the RNA activity is becoming increasingly dependent on its protein splicing factors.     

A collapsed state functions to self-chaperone RNA folding into a native ribonucleoprotein complex

Most large RNAs achieve their active, native structures only as complexes with one or more cofactor proteins. By varying the Mg(2+) concentration, the catalytic core of the bI5 group I intron RNA can be manipulated into one of three states, expanded, collapsed or native, or into balanced equilibria between these states. Under near-physiological conditions, the bI5 RNA folds rapidly to a collapsed but non-native state. Hydroxyl radical footprinting demonstrates that assembly with the CBP2 protein cofactor chases the RNA from the collapsed state to the native state. In contrast, CBP2 also binds to the RNA in the expanded state to form many non-native interactions. This structural picture is reinforced by functional splicing experiments showing that RNA in an expanded state forms a non-productive, kinetically trapped complex with CBP2. Thus, rapid folding to the collapsed state functions to self-chaperone bI5 RNA folding by preventing premature interaction with its protein cofactor. This productive, self-chaperoning role for RNA collapsed states may be especially important to avert misassembly of large multi-component RNA-protein machines in the cell.     

Near native structure in an RNA collapsed state

Many large RNAs form conformationally collapsed, but non-native, states prior to folding to the native state or assembling with protein cofactors. Although RNA collapsed states play fundamental roles in RNA folding and ribonucleoprotein assembly processes, their structures have been poorly understood. We obtained 12 high-quality structural constraints for the collapsed state formed by the catalytic core of the bI5 intron RNA using site-specific cross-linking mediated by a short-lived reactant. RNA tertiary structures in the collapsed and native states are indistinguishable, even though only the native state forms a solvent-inaccessible core. Thus, structural neighbors in the collapsed state, including several long-range tertiary interactions, are approximately as close in space as in the native state, but RNA packing is sufficiently loose or dynamic to allow access by solvent. Binding by the obligate CBP2 protein cofactor has almost no effect on structural neighbors reported by cross-linking, even though protein binding chases the RNA from the collapsed state to the native state. Protein binding thus appears to promote only the final few angstroms of RNA folding rather than mediate global conformational rearrangements in the catalytic core. The bI5 RNA collapsed state functions to self-chaperone ribonucleoprotein assembly because this conformationally restrained structure lies very near that of the native state and excludes structures that otherwise misassemble efficiently.     

Protein-dependent transition states for ribonucleoprotein assembly

Native folding and splicing by the Saccharomyces cerevisiae mitochondrial bI5 group I intron RNA is facilitated by both the S. cerevisiae CBP2 and Neurospora crassa CYT-18 protein cofactors. Both protein-bI5 RNA complexes splice at similar rates, suggesting that the RNA active site structure is similar in both ribonucleoproteins. In contrast, the two proteins assemble with the bI5 RNA by distinct mechanisms and bind opposing, but partially overlapping, sides of the group I intron catalytic core. Assembly with CBP2 is limited by a slow, unimolecular RNA folding step characterized by a negligible activation enthalpy. We show that assembly with CYT-18 shows four distinctive features. (1) CYT-18 binds stably to the bI5 RNA at the diffusion controlled limit, but assembly to a catalytically active RNA structure is still limited by RNA folding, as visualized directly using time-resolved footprinting. (2) This mechanism of rapid stable protein binding followed by subsequent assembly steps has a distinctive kinetic signature: the apparent ratio of k(off) to k(on), determined in a partitioning experiment, differs from the equilibrium K(d) by a large factor. (3) Assembly with CYT-18 is characterized by a large activation enthalpy, consistent with a rate limiting conformational rearrangement. (4) Because assembly from the kinetically trapped state is faster at elevated temperature, we can identify conditions where CYT-18 accelerates (catalyzes) bI5 RNA folding relative to assembly with CBP2.     

DNA polymerization catalysed by a group II intron RNA in vitro.

The excised group II intron bI1 from Saccharomyces cerevisiae can act as a ribozyme catalysing various chemical reactions with different substrate RNAs in vitro . Recently, we have described an editing-like RNA polymerization reaction catalysed by the bI1 intron lariat that proceeds in the 3'-->5'direction. Here we show that the bI1 lariat RNA can also catalyse successive deoxyribonucleotide polymerization reactions on exogenous substrate molecules. The basic mechanism of the reaction involved interacting cycles between an alternative version of partial reverse splicing (lariat charging) and canonical forward splicing (lariat discharging by exon ligation). With an overall chain growth in the 3'-->5' direction, the 5' exon RNAs (IBS1dN) were elongated by successive insertion of deoxyribonucleotides derived from single deoxyribonucleotide substitutions (dA, dG, dC or dT). All four deoxyribonucleotides were used as substrates, although with different efficiencies. Our findings extend the catalytic repertoire of group II intron RNAs not only by a novel DNA polymerization activity, but also by a DNA-DNA ligation capacity, supporting the idea that ribozymes might have been part of the first primordial polymerization machinery for both RNA and DNA.     

Architecture of a gamma retroviral genomic RNA dimer

Retroviral genomes contain two sense-strand RNAs that are noncovalently linked at their 5' ends, forming a dimer. Establishing a structure for this dimer is an obligatory first step toward understanding the fundamental role of the dimeric RNA in retroviral biology. We developed a secondary structure model for the minimal dimerization active sequence (MiDAS) for the Moloney murine sarcoma virus in the final dimer state using selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE). In this model, two self-complementary, or palindromic, sequences (PAL1 and PAL2) form extended intermolecular duplexes of 10 and 16 base pairs, respectively. The monomeric starting state was shown previously to contain a flexible domain in which nucleotides do not form stable interactions with other parts of the RNA. In the final dimer state, portions of this initial flexible domain form stable base pairs, while previously base-paired elements lie in a new flexible domain. Thus, partially overlapping and structurally well-defined flexible domains are prominent features of both monomer and dimer states. We then used hydroxyl radical cleavage experiments to characterize the global architecture of the dimer state. Extensive regions, including portions of both PAL1 and PAL2, are occluded from solvent-based cleavage indicating that the MiDAS domain does not function simply as a collection of autonomous secondary structure elements. Instead, the retroviral dimerization domain adopts a compact architecture characterized by close packing of its constituent helices.     

The Mrs1 Splicing Factor Binds the bI3 Group I Intron at Each of Two Tetraloop-Receptor Motifs

Most large ribozymes require protein cofactors in order to function efficiently. The yeast mitochondrial bI3 group I intron requires two proteins for efficient splicing, Mrs1 and the bI3 maturase. Mrs1 has evolved from DNA junction resolvases to function as an RNA cofactor for at least two group I introns; however, the RNA binding site and the mechanism by which Mrs1 facilitates splicing were unknown. Here we use high-throughput RNA structure analysis to show that Mrs1 binds a ubiquitous RNA tertiary structure motif, the GNRA tetraloop-receptor interaction, at two sites in the bI3 RNA. Mrs1 also interacts at similar tetraloop-receptor elements, as well as other structures, in the self-folding Azoarcus group I intron and in the RNase P enzyme. Thus, Mrs1 recognizes general features found in the tetraloop-receptor motif. Identification of the two Mrs1 binding sites now makes it possible to create a model of the complete six-component bI3 ribonucleoprotein. All protein cofactors bind at the periphery of the RNA such that every long-range RNA tertiary interaction is stabilized by protein binding, involving either Mrs1 or the bI3 maturase. This work emphasizes the strong evolutionary pressure to bolster RNA tertiary structure with RNA-binding interactions as seen in the ribosome, spliceosome, and other large RNA machines.     

Structural basis for the self-chaperoning function of an RNA collapsed state

Prior to folding to a native functional structure, many large RNAs form conformationally collapsed states. Formation of the near-native collapsed state for the bI5 group I intron RNA plays an obligatory role in self-chaperoning assembly with its CBP2 protein cofactor by preventing formation of stable, misassembled complexes. We show that the collapsed state is essential because CBP2 assembles indiscriminately with the bI5 RNA in any folding state to form long-lived complexes. The most stable protein interaction site in the expanded state-CBP2 complex overlaps, but is not identical to, the native site. Folding to the collapsed state circumvents two distinct misassembly events: inhibitory binding by multiple equivalents of CBP2 and formation of bridged complexes in which CBP2 straddles cognate and noncognate RNAs. Strikingly, protein-bound sites in the expanded state RNA complex are almost the inverse of native RNA-RNA and RNA-protein interactions, indicating that folding to the collapsed state significantly reduces the fraction of RNA surfaces accessible for misassembly. The self-chaperoning function for the bI5 collapsed state is likely to be conserved in other ribonucleoproteins where a protein cofactor binds tightly at a simple RNA substructure or has an RNA binding surface composed of multiple functional sites.     

Two distinct binding modes of a protein cofactor with its target RNA

Like most cellular RNA enzymes, the bI5 group I intron requires binding by a protein cofactor to fold correctly. Here, we use single-molecule approaches to monitor the structural dynamics of the bI5 RNA in real time as it assembles with its CBP2 protein cofactor. These experiments show that CBP2 binds to the target RNA in two distinct modes with apparently opposite effects: a "non-specific" mode that forms rapidly and induces large conformational fluctuations in the RNA, and a "specific" mode that forms slowly and stabilizes the native RNA structure. The bI5 RNA folds though multiple pathways toward the native state, typically traversing dynamic intermediate states induced by non-specific binding of CBP2. These results suggest that the protein cofactor-assisted RNA folding involves sequential non-specific and specific protein-RNA interactions. The non-specific interaction potentially increases the local concentration of CBP2 and the number of conformational states accessible to the RNA, which may promote the formation of specific RNA-protein interactions.     

The bI4 group I intron binds directly to both its protein splicing partners, a tRNA synthetase and maturase, to facilitate RNA splicing activity

The imported mitochondrial leucyl-tRNA synthetase (NAM2p) and a mitochondrial-expressed intron-encoded maturase protein are required for splicing the fourth intron (bI4) of the yeast cob gene, which expresses an electron transfer protein that is essential to respiration. However, the role of the tRNA synthetase, as well as the function of the bI4 maturase, remain unclear. As a first step towards elucidating the mechanistic role of these protein splicing factors in this group I intron splicing reaction, we tested the hypothesis that both leucyl-tRNA synthetase and bI4 maturase interact directly with the bI4 intron. We developed a yeast three-hybrid system and determined that both the tRNA synthetase and bI4 maturase can bind directly and independently via RNA-protein interactions to the large bI4 group I intron. We also showed, using modified two-hybrid and three-hybrid assays, that the bI4 intron bridges interactions between the two protein splicing partners. In the presence of either the bI4 maturase or the Leu-tRNA synthetase, bI4 intron transcribed recombinantly with flanking exons in the yeast nucleus exhibited splicing activity. These data combined with previous genetic results are consistent with a novel model for a ternary splicing complex (two protein: one RNA) in which both protein splicing partners bind directly to the bI4 intron and facilitate its self-splicing activity.     

RNA secondary structure and compensatory evolution

The classic concept of epistatic fitness interactions between genes has been extended to study interactions within gene regions, especially between nucleotides that are important in maintaining pre-mRNA/mRNA secondary structures. It is shown that the majority of linkage disequilibria found within the Drosophila Adh gene are likely to be caused by epistatic selection operating on RNA secondary structures. A recently proposed method of RNA secondary structure prediction based on DNA sequence comparisons is reviewed and applied to several types of RNAs, including tRNA, rRNA, and mRNA. The patterns of covariation in these RNAs are analyzed based on Kimura's compensatory evolution model. The results suggest that this model describes the substitution process in the pairing regions (helices) of RNA secondary structures well when the helices are evolutionarily conserved and thermodynamically stable, but fails in some other cases. Epistatic selection maintaining pre-mRNA/mRNA secondary structures is compared to weak selective forces that determine features such as base composition and synonymous codon usage. The relationships among these forces and their relative strengths are addressed. Finally, our mutagenesis experiments using the Drosophila Adh locus are reviewed. These experiments analyze long-range compensatory interactions between the 5' and 3' ends of Adh mRNA, the different constraints on secondary structures in introns and exons, and the possible role of secondary structures in RNA splicing.     

Variation in sequence and RNA editing within core domains of mitochondrial group II introns among plants

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.     

A collapsed non-native RNA folding state

At physiological Mg2+ concentrations, the catalytic core of the bI5 group I intron does not fold into its native structure. In contrast, as judged by the global size, this RNA undergoes structural collapse at Mg 2+ concentrations much lower than required to drive folding of the RNA completely to the native state. The bI5 RNA therefore exists in equilibrium between expanded and collapsed non-native states. The activation energy of RNA folding from the collapsed state to the native state is negligible and the reaction is not accelerated by the addition of urea. This collapsed state is thus distinct from the kinetic traps observed during folding of other large RNAs. The collapsed non-native state forms readily in the case of bI5 RNA and may exist generically prior to assembly of other ribonucleoprotein holoenzymes, such as the ribosome.     

Mapping divalent metal ion binding sites in a group II intron by Mn(2+)- and Zn(2+)-induced site-specific RNA cleavage.

The function of group II introns depends on positively charged divalent metal ions that stabilize the ribozyme structure and may be directly involved in catalysis. We investigated Mn2+- and Zn2+-induced site-specific RNA cleavage to identify metal ions that fit into binding pockets within the structurally conserved bI1 group II intron domains (DI-DVI), which might fulfill essential roles in intron function. Ten cleavage sites were identified in DI, two sites in DIII and two in DVI. All cleavage sites are located in the center or close to single-stranded and flexible RNA structures. Strand scissions mediated by Mn2+/Zn2+ are competed for by Mg2+, indicating the existence of Mg2+ binding pockets in physical proximity to the observed Mn2+-/Zn2+-induced cleavage positions. To distinguish between metal ions with a role in structure stabilization and those that play a more specific and critical role in the catalytic process of intron splicing, we combined structural and functional assays, comparing wild-type precursor and multiple splicing-deficient mutants. We identified six regions with binding pockets for Mg2+ ions presumably playing an important role in bI1 structure stabilization. Remarkably, assays with DI deletions and branch point mutants revealed the existence of one Mg2+ binding pocket near the branching A, which is involved in first-step catalysis. This pocket formation depends on precise interaction between the branching nucleotide and the 5' splice site, but does not require exon-binding site 1/intron binding site 1 interaction. This Mg2+ ion might support the correct placing of the branching A into the 'first-step active site'.