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The internal loop structure of the active stem-loop I is a tertiary interaction motif . ( a ) Sequence and secondary structure of the pattern searched by MC - SEARCH and of helix 44 of 16S rRNA of T. thermophilus (40) . ( b ) A superposition between helix 44 (PDB ID code 1FJG; pastel colors) and the minimized average structure of SL1 Ј (darker colors) was obtained by minimizing the rmsd for heavy atoms of the fi ve residues in the internal loop (1.40 Å ). Also shown are residues in helix 13 of 16S rRNA, which form a tertiary interaction with helix 44 . ( c ) Summary of the base-pairing and stacking interactions in helices 13 and 44 and of tertiary contacts between them. Solid and dashed black lines indicate base pairs with two hydrogen bonds (either Watson – Crick or sheared G-A) and one hydrogen bond, respectively. Black rectangles indicate base stacking. Red spheres indicate riboses involved in ribose – ribose contacts (red dashed lines). The two adenines A1433 and A1434 participate in A-minor motifs (green dashed lines).
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Substrate cleavage by the Neurospora Varkud satellite (VS) ribozyme involves a structural change in the stem-loop I substrate from an inactive to an active conformation. We have determined the NMR solution structure of a mutant stem-loop I that mimics the active conformation of the cleavage site internal loop. This structure shares many similaritie...
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... Occurrences of the Active Internal Loop Fold. To investigate whether the SL1 internal loop fold exists in other RNAs, we used an automated program MC-SEARCH (P.G. and F.M., un- published data) to search for RNA structure patterns in the PDB (31). The pattern searched contained the sequence of the SL1 internal loop flanked by closing base pairs (Fig. 5a). MC-SEARCH found several occurrences of this pattern (Fig. 5a) in the database, consisting of multiple structures of two different helical domains of rRNA. One is from helix 44 of 16S rRNA found in the small ribosomal subunit of Thermus thermophilus (40) (Fig. 5). The other one is from helix 25 of 23S rRNA found in the large ribosomal ...
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... whether the SL1 internal loop fold exists in other RNAs, we used an automated program MC-SEARCH (P.G. and F.M., un- published data) to search for RNA structure patterns in the PDB (31). The pattern searched contained the sequence of the SL1 internal loop flanked by closing base pairs (Fig. 5a). MC-SEARCH found several occurrences of this pattern (Fig. 5a) in the database, consisting of multiple structures of two different helical domains of rRNA. One is from helix 44 of 16S rRNA found in the small ribosomal subunit of Thermus thermophilus (40) (Fig. 5). The other one is from helix 25 of 23S rRNA found in the large ribosomal subunit of Haloarcula marismortui (41) (Fig. 8, which is ...
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... contained the sequence of the SL1 internal loop flanked by closing base pairs (Fig. 5a). MC-SEARCH found several occurrences of this pattern (Fig. 5a) in the database, consisting of multiple structures of two different helical domains of rRNA. One is from helix 44 of 16S rRNA found in the small ribosomal subunit of Thermus thermophilus (40) (Fig. 5). The other one is from helix 25 of 23S rRNA found in the large ribosomal subunit of Haloarcula marismortui (41) (Fig. 8, which is published as supporting information on the PNAS web site). Surprisingly, even though no hydrogen bonding or stacking interactions were specified in the internal loop of the pattern searched, the RNAs found ...
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... the PNAS web site). Surprisingly, even though no hydrogen bonding or stacking interactions were specified in the internal loop of the pattern searched, the RNAs found are very similar in structure to the internal loop of SL1. The sequence of helix 44 of 16S rRNA matches exactly the sequence of the internal loop of SL1 and its closing base pairs (Fig. 5a). Heavy-atom superposition of internal loop residues of helix 44 of 16S rRNA [PDB ID code 1FJG (40)] and SL1 yields a rmsd of 1.40 Å (Fig. 5b). The main difference between these structures is in the shared sheared G-A base pairs; in helix 44, A1433 is displaced away from G1467 such that there is only one potential hydrogen bond between ...
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... searched, the RNAs found are very similar in structure to the internal loop of SL1. The sequence of helix 44 of 16S rRNA matches exactly the sequence of the internal loop of SL1 and its closing base pairs (Fig. 5a). Heavy-atom superposition of internal loop residues of helix 44 of 16S rRNA [PDB ID code 1FJG (40)] and SL1 yields a rmsd of 1.40 Å (Fig. 5b). The main difference between these structures is in the shared sheared G-A base pairs; in helix 44, A1433 is displaced away from G1467 such that there is only one potential hydrogen bond between its base and the base of G1467 (Fig. ...
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... of internal loop residues of helix 44 of 16S rRNA [PDB ID code 1FJG (40)] and SL1 yields a rmsd of 1.40 Å (Fig. 5b). The main difference between these structures is in the shared sheared G-A base pairs; in helix 44, A1433 is displaced away from G1467 such that there is only one potential hydrogen bond between its base and the base of G1467 (Fig. ...
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... the minor grooves of helix 44 of 16S rRNA and helix 25 of 23S rRNA both participate in long-range tertiary interactions termed canonical ribose zippers (Figs. 5 and 8) (42). In 16S rRNA of T. thermophilus, the internal loop of helix 44 interacts with the stem of helix 13 through multiple ribose-ribose contacts, and the two adenines, A1433 and A1434, participate in A-minor motifs with C335 and G319-C334, respectively (Fig. 5c) (43). This ribose zipper is similar to that formed by the GAAA tetraloop ...
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... participate in long-range tertiary interactions termed canonical ribose zippers (Figs. 5 and 8) (42). In 16S rRNA of T. thermophilus, the internal loop of helix 44 interacts with the stem of helix 13 through multiple ribose-ribose contacts, and the two adenines, A1433 and A1434, participate in A-minor motifs with C335 and G319-C334, respectively (Fig. 5c) (43). This ribose zipper is similar to that formed by the GAAA tetraloop and tetraloop receptor, where two consecutive ad- enines stacked on a sheared G-A base pair participate in A-minor motifs (42,44,45). In addition, the base stacking PNAS June 10, 2003 vol. 100 no. 12 7007 pattern in helix 44 of 16S rRNA (43) and in the internal ...
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... helix II and helix VI (8,46,47), which contains the proposed active site (38,46,(48)(49)(50). Substrate binding to the catalytic domain of the VS ribozyme is facilitated by formation of the active stem-loop I conformation (8,51). Here, we have shown that this active conformation functions as a tertiary ribose zipper motif in 16S and 23S rRNAs (Fig. 5). Analysis of stem-loop ribose zippers in rRNA structures indicates that adenines are favored in the loop (42); a similar preference for adenine residues at positions 621 and 622 in the stem-loop I of the VS ribozyme has been demonstrated from in vitro selection experiments (11). In the active stem-loop I conformation, A621 and A622 are ...
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... zippers in rRNA structures indicates that adenines are favored in the loop (42); a similar preference for adenine residues at positions 621 and 622 in the stem-loop I of the VS ribozyme has been demonstrated from in vitro selection experiments (11). In the active stem-loop I conformation, A621 and A622 are well positioned to form A-minor motifs (Fig. 5b), whereas in the inactive stem-loop I, the A622 -C637 base pair (Fig. 3) would hinder such interaction. Based on these similar- ities in sequence and structure, we speculate that the active conformation of the VS ribozyme stem-loop I internal loop forms a ribose zipper with either helix II or VI of the catalytic ...
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The Neurospora VS ribozyme is a small nucleolytic ribozyme with unique primary, secondary and global tertiary structures, which displays
mechanistic similarities to the hairpin ribozyme. Here, we determined the high-resolution NMR structure of a stem–loop VI
fragment containing the A730 internal loop, which forms part of the active site. In the pre...
Citations
... These mismatched regions are thought to mediate important intermolecular interactions, including protein recognition [45]- [51] and define the global RNA structure [52]- [55]. Internal loops have also been shown to be important for RNA-mediated processes like ribozyme cleavage [56]- [58], RNA conformational changes [59]- [61], and RNA processing [62]- [65]. ...
MicroRNAs (miRNAs) are important regulators of post-transcriptional gene expression. Mature miRNAs are generated from longer transcripts (primary, pri- and precursor, pre-miRNAs) through a series of highly coordinated enzymatic processing steps. The sequence and structure of these pri- and pre-miRNAs play important roles in controlling their processing. Both pri- and pre-miRNAs adopt hairpin structures with imperfect base pairing in the helical stem. Here, we investigated the role of three base pair mismatches (A∙A, G∙A, and C∙A) present in pre-miRNA-31. Using a combination of NMR spectroscopy and thermal denaturation, we found that nucleotides within the three base pair mismatches displayed unique structural properties, including varying dynamics and sensitivity to solution pH. These studies deepen our understanding of how the physical and chemical properties of base pair mismatches influence RNA structural stability.
... Previous structural studies on the Neurospora VS ribozyme have determined the crystal structure of a domain-swapped dimer by X-ray crystallography (43,44) and solution structures of several isolated VS ribozyme subdomains by NMR spectroscopy (40,41,45,46,70,73). The present study combines the high-resolution NMR structures of subdomains to determine the solution structure of a minimal trans VS ribozyme in its functional monomeric state. ...
The divide-and-conquer strategy is commonly used for protein structure determination, but its applications to high-resolution structure determination of RNAs have been limited. Here, we introduce an integrative approach based on the divide-and-conquer strategy that was undertaken to determine the solution structure of an RNA model system, the Neurospora VS ribozyme. NMR and SAXS studies were conducted on a minimal trans VS ribozyme as well as several isolated subdomains. A multi-step procedure was used for structure determination that first involved pairing refined NMR structures with SAXS data to obtain structural subensembles of the various subdomains. These subdomain structures were then assembled to build a large set of structural models of the ribozyme, which was subsequently filtered using SAXS data. The resulting NMR-SAXS structural ensemble shares several similarities with the reported crystal structures of the VS ribozyme. However, a local structural difference is observed that affects the global fold by shifting the relative orientation of the two three-way junctions. Thus, this finding highlights a global conformational change associated with substrate binding in the VS ribozyme that is likely critical for its enzymatic activity. Structural studies of other large RNAs should benefit from similar integrative approaches that allow conformational sampling of assembled fragments.
... The VS ribozyme undergoes major rearrengements during the folding of the active conformation that include changes in the RNA secondary structure. 125,126 As in the hairpin ribozyme, tertiary interactions are involved during the folding in the presence of divalent metal ions; one of these well-studied interactions is a magnesium-dependent loop/loop pairing between the stem-loops I and V. 127,128 In the reaction pathway of the hairpin ribozyme, several states were isolated and characterized. In the case of the VS ribozyme, the kinetic model of the reaction pathway is much more complex due to the size of the RNA with a hierarchical folding path. ...
The use of high hydrostatic pressure to investigate structure-function relationships in biomacromolecules in solution provides precise information about conformational changes and variations of the interactions between these macromolecules and the solvent, as well as the volume changes associated with their activity. The complementary use of osmotic pressure reveals quantitatively the extent and direction of the water exchanges between the macromolecules and the solvent and the number of water molecules involved in these exchanges. In this review, the chemistry of ribozymes and the influence of pressure is described. In the case of the hairpin ribozyme, pressure slowed down the self-cleavage reaction on the basis that the formation of the transition state involves a positive ΔV⧧ of activation and the release of 78 ± 4 water molecules. The self-cleaving activity of the hammerhead ribozyme is also slowed down by pressure on the basis of kinetic parameters and ΔVs comparable to those of the hairpin ribozymes. However, it appears that the solution of the hammerhead ribozyme used in this study contains two populations of molecules which differ by the values of these parameters. The results obtained in the case of small self-cleaving ribozymes containing adenine bulges are consistent with the hypothesis that these small RNAs that bind amino acids or peptides could have appeared in prebiotic chemistry under extreme conditions in deep-sea vents or hydrothermal surface sites.
... Interestingly, both loops adopt a U-turn structure. In the case of SLI, this is part of a larger seven-membered loop, though the canonical four-member UNRA sequence is adopted, with characteristic interactions (13). For SLV, the formation of a U-turn structure has been shown to be Mg 2þ dependent, along with a structural shift upon Mg 2þ binding (14,15). ...
... Interestingly, both loops adopt a U-turn structure. In the case of SLI, this is part of a larger seven-membered loop, though the canonical four-member UNRA sequence is adopted, with characteristic interactions (13). For SLV, the formation of a U-turn structure has been shown to be Mg 2þ dependent, along with a structural shift upon Mg 2þ binding (14,15). ...
Though the structure of the substrate stem loop I (SLI)-stem loop V (SLV) kissing loop junction of the Varkud Satellite ribozyme has been experimentally characterized, the dynamics of this Mg²⁺-dependent loop-loop interaction have been elusive. Specifically, each hairpin loop contains a U-turn motif, but only SLV shows a conformational shift triggered by Mg²⁺ ion association. Here, we use molecular dynamics simulations to analyze the binding and dynamics of this kissing loop junction. We show that SLV acts as a scaffold, providing stability to the junction. Mg²⁺ ions associate with SLV when it is part of the junction in a manner similar to when it is unbound, but there is no specificity in Mg²⁺ binding for the SLI loop. This suggests that the entropic penalty of ordering the larger SLI is too high, allowing SLV to act as a scaffold for multiple substrate loop sequences.
... 40 Unshifted and Shifted States of G 638 Loop NMR structures of the SLI substrate were determined to characterize both the unshifted (inactive) and shifted (active) states of the substrate cleavage site internal loop, termed here the G 638 loop. [16][17][18] Two individual structures were determined for the unshifted state, one of which is illustrated in Figure 2 (a). 16,17 In both studies, the RNA stem-loop used comprises the natural internal loop sequence and the adjacent base pairs from stems Ia and Ib. ...
... At higher pH, the G 638 loop adopts a more open conformation that may facilitate the displacement of residue C 637 into stem Ib as part of the conformational change associated with formation of the I/V KLI. 17 The structure of the shifted conformation of the G 638 loop was also determined by NMR, as part of an RNA stem-loop with a nonnatural stem Ib and a GNRA tetraloop that stabilize the shifted state ( Figure 2(b)). 18 As expected, this RNA was cleaved when incubated with a trans VS ribozyme, although at a lower rate than a wild-type sequence given that the I/V KLI could not form due to the terminal loop modification. 18 The NMR structure revealed that residues G 620 and A 639 form a trans H/SE G-A base pair, as found for the unshifted state (Figure 2(a) and (b)). ...
... 18 As expected, this RNA was cleaved when incubated with a trans VS ribozyme, although at a lower rate than a wild-type sequence given that the I/V KLI could not form due to the terminal loop modification. 18 The NMR structure revealed that residues G 620 and A 639 form a trans H/SE G-A base pair, as found for the unshifted state (Figure 2(a) and (b)). [16][17][18] However, residue G 638 is positioned such that it can participate in a trans H/SE G-A base pair with either A 621 or A 622 (Figure 2(b)). ...
Despite the large number of noncoding RNAs and their importance in several biological processes, our understanding of RNA structure and dynamics at atomic resolution is still very limited. Like many other RNAs, the Neurospora Varkud satellite (VS) ribozyme performs its functions through dynamic exchange of multiple conformational states. More specifically, the VS ribozyme recognizes and cleaves its stem‐loop substrate via a mechanism that involves several structural transitions within its stem‐loop substrate. The recent publications of high‐resolution structures of the VS ribozyme, obtained by NMR spectroscopy and X‐ray crystallography, offer an opportunity to integrate the data and closely examine the structural and dynamic properties of this model RNA system. Notably, these investigations provide a valuable example of the divide‐and‐conquer strategy for structural and dynamic characterization of a large RNA, based on NMR structures of several individual subdomains. The success of this divide‐and‐conquer approach reflects the modularity of RNA architecture and the great care taken in identifying the independently‐folding modules. Together with previous biochemical and biophysical characterizations, the recent NMR and X‐ray studies provide a coherent picture into how the VS ribozyme recognizes its stem‐loop substrate. Such in‐depth characterization of this RNA enzyme will serve as a model for future structural and engineering studies of dynamic RNAs and may be particularly useful in planning divide‐and‐conquer investigations. WIREs RNA 2017, 8:e1421. doi: 10.1002/wrna.1421
For further resources related to this article, please visit the WIREs website.
... The SLI-SLV interaction facilitates the docking of SLI with SLVI, thereby forming the active site [65,67,68]. The SLI-SLV kissing loop NMR structure has been solved [69][70][71] and supporting isothermal titration calorimetry experiments demonstrate that the SLI-SLV kissing-loop interaction is a major thermodynamic barrier and, therefore, is likely regulating ribozyme activity [61]. The formation of the SLI-SLV interaction relies upon divalent metal cations; however, there is no evidence for their direct involvement in catalysis. ...
Self-cleaving ribozymes were discovered 30 years ago, but their biological distribution and catalytic mechanisms are only beginning to be defined. Each ribozyme family is defined by a distinct structure, with unique active sites accelerating the same transesterification reaction across the families. Biochemical studies show that general acid-base catalysis is the most common mechanism of self-cleavage, but metal ions and metabolites can be used as cofactors. Ribozymes have been discovered in highly diverse genomic contexts throughout nature, from viroids to vertebrates. Their biological roles include self-scission during rolling-circle replication of RNA genomes, co-transcriptional processing of retrotransposons, and metabolite-dependent gene expression regulation in bacteria. Other examples, including highly conserved mammalian ribozymes, suggest that many new biological roles are yet to be discovered.
... In the NMR structure of stem-loop 1 in isolation [39][40][41] , A621 and A622 remain inside the loop whereas in the crystal structure these nucleotides protrude from the helical stack to make specific interactions with the ribozyme core (Supplementary Fig. 9e,h). Remodeling also occurs in loops A and B of the hairpin ribozyme, as suggested by comparison of the crystal structure of the complete ribozyme with the NMR structure of the isolated loops 42,43 . ...
The Varkud satellite (VS) ribozyme mediates rolling-circle replication of a plasmid found in the Neurospora mitochondrion. We report crystal structures of this ribozyme from Neurospora intermedia at 3.1 Å resolution, which revealed an intertwined dimer formed by an exchange of substrate helices. In each protomer, an arrangement of three-way helical junctions organizes seven helices into a global fold that creates a docking site for the substrate helix of the other protomer, resulting in the formation of two active sites in trans. This mode of RNA-RNA association resembles the process of domain swapping in proteins and has implications for RNA regulation and evolution. Within each active site, adenine and guanine nucleobases abut the scissile phosphate, poised to serve direct roles in catalysis. Similarities to the active sites of the hairpin and hammerhead ribozymes highlight the functional importance of active-site features, underscore the ability of RNA to access functional architectures from distant regions of sequence space, and suggest convergent evolution.
... The VS ribozyme is a moderately sized ribozyme with a rich structure (Figure 4). The full 3D structure of the ribozyme is not yet available, but NMR has been extensively employed to study important localities of the ribozyme [83,[86][87][88][89][90][91][92][93][94]. ...
The notion of fitness landscapes, a map between genotype and fitness, was proposed more than 80 years ago. For most of this time data was only available for a few alleles, and thus we had only a restricted view of the whole fitness landscape. Recently, advances in genetics and molecular biology allow a more detailed view of them. Here we review experimental and theoretical studies of fitness landscapes of functional RNAs, especially aptamers and ribozymes. We find that RNA structures can be divided into critical structures, connecting structures, neutral structures and forbidden structures. Such characterisation, coupled with theoretical sequence-to-structure predictions, allows us to construct the whole fitness landscape. Fitness landscapes then can be used to study evolution, and in our case the development of the RNA world.
... Although no complete high-resolution structure has been reported for the VS ribozyme, low-resolution models have been derived from biochemical, fluorescence resonance energy transfer (FRET), and small-angle X-ray scattering (SAXS) studies that provide insights into its global structure (Hiley and Collins 2001;Lafontaine et al. 2001aLafontaine et al. , 2002Lipfert et al. 2008). Moreover, several high-resolution NMR structures of isolated subdomains of the Neurospora VS ribozyme have been determined, including the structures of the SLI substrate in its inactive (Michiels et al. 2000;Flinders and Dieckmann 2001) and active forms (Hoffmann et al. 2003), the terminal loop of SLV in the presence and absence of magnesium ions (Mg 2+ ) (Campbell and Legault 2005;Campbell et al. 2006), the I/V kissing-loop interaction (Bouchard and Legault 2014b), the A730 loop of SLVI (Flinders and Dieckmann 2004;Desjardins et al. 2011;Bonneau and Legault 2014a), and the III-IV-V three-way junction (Bonneau and Legault 2014b). Our laboratory has contributed significantly to determining NMR structures of VS ribozyme subdomains with the goal of defining a complete high-resolution solution structure of the Neurospora VS ribozyme. ...
... A three-dimensional model of a VS ribozyme substrate/ribozyme complex ( Fig. 7A; Lacroix-Labonté et al. 2012) was generated by assembly of NMR structures of individual subdomains in PyMOL based on superposition of overlapping helical segments. The following NMR structures of isolated subdomains were used: the cleavage site internal loop of the SLI substrate in its active form (PDB ID code 1OW9) (Hoffmann et al. 2003), the terminal loop of SLV in the presence of Mg 2+ (PDB ID code 1YN1) (Campbell et al. 2006), the I/V kissing-loop interaction (PDB ID code 2MI0) (Bouchard and Legault 2014b), the A730 loop of SLVI (PDB ID code 2L5Z) (Desjardins et al. 2011), the III-IV-V junction (PDB ID code 2MTJ) (Bonneau and Legault 2014b) and the II-III-VI junction (PDB ID code 2N3Q). The initial model was then minimized with the molecular dynamics package GROMACS (Pronk et al. 2013) and the Amber99SB force field with the ParmBSC0 nucleic acid parameters and using explicit aqueous solvent (Hornak et al. 2006;Perez et al. 2007;Guy et al. 2012). ...
As part of an effort to structurally characterize the complete Neurospora VS ribozyme, NMR solution structures of several subdomains have been previously determined, including the internal loops of domains I and VI, the I/V kissing-loop interaction and the III-IV-V junction. Here, we expand this work by determining the NMR structure of a 62-nucleotide RNA (J236) that encompasses the VS ribozyme II-III-VI three-way junction and its adjoining stems. In addition, we localize Mg(2+)-binding sites within this structure using Mn(2+)-induced paramagnetic relaxation enhancement. The NMR structure of the J236 RNA displays a family C topology with a compact core stabilized by continuous stacking of stems II and III, a cis WC/WC G•A base pair, two base triples and two Mg(2+) ions. Moreover, it reveals a remote tertiary interaction between the adenine bulges of stems II and VI. Additional NMR studies demonstrate that both this bulge-bulge interaction and Mg(2+) ions are critical for the stable folding of the II-III-VI junction. The NMR structure of the J236 RNA is consistent with biochemical studies on the complete VS ribozyme, but not with biophysical studies performed with a minimal II-III-VI junction that does not contain the II-VI bulge-bulge interaction. Together with previous NMR studies, our findings provide important new insights into the three-dimensional architecture of this unique ribozyme.
© 2015 Bonneau et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.
