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Insights into RNA structure and dynamics from recent NMR and X‐ray studies of the Neurospora Varkud satellite ribozyme

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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 This article is categorized under: RNA Structure and Dynamics > RNA Structure, Dynamics, and Chemistry RNA-Based Catalysis > RNA-Mediated Cleavage
The Neurospora VS ribozyme. Primary and secondary structures of the natural cis‐cleaving VS ribozyme (residues 617–783) determined from mutagenesis and chemical probing studies. Recognition of the substrate (stem‐loop I or SLI) involves a KLI with stem‐loop V (SLV), an activating conformation change (left) and a loop–loop interaction between the G638 and A756 internal loops to form the active site. Helical domains are color‐coded and identified with roman numerals (I–VI). The scissile phosphate is depicted by a red dot.
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Formation of the active site in the VS ribozyme. (a) Remodeling of the A756 loop (left) and G638 loop (right) upon formation of the active site as seen from the superposition of the lowest‐energy NMR structures of the free loops [in gray; PDB codes 2L5Z (left) and 1OW9 (right)] onto those forming the active site in the crystal structure (in color; PDB code 4R4P). (b) Local environment at the active site (PDB code 4R4P). (c) Key residues at the active site of the G638A variant (left; PDB code 4R4V) and the A756G variant (right; PDB code 4R4P). The representation highlights the atoms that are expected to adopt an inline geometry in the transition state, namely the 2′‐oxygen (gold sphere), scissile P (red), and 5′‐oxygen (gold sphere) that rather form angles of 97° (left) and 129° (right). The N1 atoms of G638 and A638 are respectively 5.7 Å (left) and 4.9 Å (right) away from the 2′‐oxygen nucleophile (dashed line), whereas the N1 atoms of A756 and G756 are respectively 4.2 Å (left) and 4.0 Å (right) away from the 5′‐oxygen leaving group (dashed line). (Adapted with permission from Ref 34. Macmillan Publishers Ltd: Nature Chemical Biology)
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Comparative analysis of the crystal and NMR structures in terms of the structural features that direct the folding of the VS ribozyme. A general view of the crystal structure (PDB code 4R4V; left) and specific characteristic features (right) are provided for (a) the I/V kissing‐loop interaction (residues 623–637 and 692–704), (b) the III–IV–V junction (residues 661–670, 681–691, and 705–717), and (c) the II–III–VI junction (residues 649–662, 716–729, and 758–774). Each specific feature is displayed as a side‐by‐side comparison between the X‐ray crystallography structure (PDB code 4R4V) on the left and a superposition of the 10 lowest‐energy NMR structures [PDB codes 2MI0 (a), 2MTJ (b), and 2N3R (c)] on the right. Divalent metal ions that are common to both the NMR and X‐ray structures are shown as black spheres.
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Structural overview of the closed state of the VS ribozyme captured by the crystal structure of a dimeric form. (a) Proposed secondary structure (as in Figure ) of the two cis ribozyme sequences used for crystal structure determination, a G638A and an A756G variant. Of note, the secondary structure was defined for a smaller RNA sequence where the I–II–VII junction and stem VII were not present. (b) Simplified global architecture of the dimeric form reflecting the relative orientation of helical domains and showing the center of two‐fold rotational symmetry. (c) Cartoon representation of the crystal structure of the G638A variant (PDB code 4R4V) shown in a simplified trans form with one entire protomer and the SLI substrate of the partner protomer. (d) Tertiary structure schematics (as in Figure ) of the simplified trans form. Due to constraints inherent to this type of representation, the proximity of the G638 and A756 loops is not depicted here. (Adapted with permission from Ref 34. Copyright Macmillan Publishers Ltd: Nature Chemical Biology)
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Structural overview of the open state of the VS ribozyme captured by the NMR‐based model of a trans ribozyme in complex with a preshifted stem‐loop substrate. (a) Proposed secondary structure of the complex, which was previously characterized kinetically and used to identify important subdomains for NMR structural determination (shaded gray boxes). (b) Simplified global architecture reflecting the relative orientation of helical domains. (c) Cartoon representation of the three‐dimensional NMR model. (d) Tertiary structure schematics (as in Figure ) based on high‐resolution NMR structures of isolated subdomains. Other legend details: In panels (a) and (d), nonnatural nucleotides are in lowercase. Also, base pairs of the proposed secondary structure are shown as a solid line when compatible with the structure shown in panel (c) and as a dashed line when not. A uniform color scheme is used for helical domains, and the scissile phosphate is depicted by a red dot or sphere.
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NMR structures of the VS ribozyme (a) III–IV–V(Adapted with permission from Ref 28. Copyrights 2014 American Chemical Society). and (b) II–III–VI three‐way junctions. From left to right: proposed secondary structures of the RNA subdomain used for NMR studies; schematic representation (as in Figure ) of the tertiary structure derived from the NMR structures; cartoon representation of the lowest‐energy NMR structure determined in the presence of Mg2+ ions; and of the lowest‐energy NMR structure determined in the presence of Mg2+ ions and using RNA‐metal restraints to localize hydrated Mg2+ ions (black spheres).
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NMR structures of an SLVI subdomain containing the A756 loop. Two NMR structures were obtained in the presence of Mg2+ ions (PDB code 2L5Z and 2MIS). The NMR structure with the PDB code 2MIS was determined with RNA‐metal restraints to localize hexahydrated Mn2+ ions (black spheres). A structural schematic (as in Figure ) is available alongside the cartoon representation of the lowest‐energy NMR structures to provide the sequence and base‐pairing interactions present in both structures. (Adapted from articles that appeared in Oxford University Press and ACS publications).
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Model for substrate recognition and activation by the VS ribozyme based on NMR data. Structural schematics (as in Figure ) are used to illustrate the unbound states of SLI and SLV (left), a hypothetical state in which the two loops initiate their contact (middle) and a final bound state (right). Base pairs of stem Ib, the KLI, and stem V are shaded in green, cyan, and pink, respectively.
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NMR structures of VS ribozyme subdomains determined to characterize substrate recognition and activation. (a) NMR structure of an SLI substrate with the G638 loop in the unshifted conformation (PDB code 1HWQ). (b) NMR structure of an SLI substrate with the G638 loop in the shifted conformation (PDB code 1OW9). The gray spheres on the structural schematics illustrate Mg2+‐binding sites near the 5′‐AG‐3′ strand of a 5′‐AG‐3′/5′‐CG‐3′ motif that are consistent with NMR chemical shift perturbations. (Adapted with permission from Ref 18. Copyright 2003 National Academy of Sciences). (c) NMR structures of the SLV subdomain of the VS ribozyme obtained in the absence of Mg2+ ions (PDB code 1TBK) and in the presence of Mg2+ ions (PDB code 1YN1 and 1YN2). The NMR structure with the PDB code 1YN2 was determined with RNA‐metal restraints to localize hexahydrated Mn2+ ions (black spheres). (Adapted with permission. Copyrights 2005 and 2006 American Chemical Society). (d) NMR structure of an SLI/SLV complex (PDB code 2MI0). In panels (a–d), a structural schematic is available alongside a cartoon representation of the NMR structure to illustrate the sequence and base‐pairing interactions present in the structure(s): the dashed boxes enclose the sequences derived from the VS ribozyme; specific residues are colored as in the NMR structure representations; and base pairs are depicted using the Leontis–Westhof notation in either black (≥2 hydrogen bonds) or gray (1 hydrogen bond).
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