Smaller, faster ribozymes reveal the catalytic core of Neurospora VS RNA

Rastogi, Toolika; Collins, Richard A.

doi:10.1006/jmbi.1997.1623

Cited by 46 publications

(59 citation statements)

References 29 publications

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“…This loop V substitution significantly reduced the self-cleavage activity of H3 (H3⌬k; Fig. 1 A), consistent with the previously observed effect of this substitution in the G11 construct (26). Self-cleavage of H3⌬k was about 10,000-fold slower than H3, barely detectable above background uncatalyzed cleavage (see Fig.…”

Section: Chemical Modification Reveals the Kissing Interaction Is Reqsupporting

confidence: 90%

Intramolecular secondary structure rearrangement by the kissing interaction of the Neurospora VS ribozyme

Andersen

Collins

2001

Proc. Natl. Acad. Sci. U.S.A.

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Kissing interactions in RNA are formed when bases between two hairpin loops pair. Intra-and intermolecular kissing interactions are important in forming the tertiary or quaternary structure of many RNAs. Self-cleavage of the wild-type Varkud satellite (VS) ribozyme requires a kissing interaction between the hairpin loops of stemloops I and V. In addition, self-cleavage requires a rearrangement of several base pairs at the base of stem I. We show that the kissing interaction is necessary for the secondary structure rearrangement of wild-type stem-loop I. Surprisingly, isolated stem-loop V in the absence of the rest of the ribozyme is sufficient to rearrange the secondary structure of isolated stem-loop I. In contrast to kissing interactions in other RNAs that are either confined to the loops or culminate in an extended intermolecular duplex, the VS kissing interaction causes changes in intramolecular base pairs within the target stem-loop.R NA kissing interactions, also called loop-loop pseudoknots, occur when the unpaired nucleotides in one hairpin loop base pair with the unpaired nucleotides in another hairpin loop (1). When the hairpin loops are located on separate RNA molecules, their intermolecular interaction is called a kissing complex. These interactions generally form between stem-loops containing extensive complementarity; however, stable complexes have been observed containing only two intermolecular Watson-Crick base pairs (2).Intramolecular kissing interactions are observed in the native structures of a variety of RNAs including Varkud satellite (VS) RNA, tRNA, and group I introns (3-6). These kissing interactions contribute to the assembly and stabilization of their respective RNA structures by joining and orienting helices. Kissing interactions may stabilize both native and nonnative interactions during tertiary folding, which can affect the rate at which the native structure is formed (7). Kissing interactions often form distorted structures that can serve as recognition sites for proteins, RNAs, metal ions, or other ligands (8-10). As a result, kissing interactions contribute to the stability of an RNA structure by affecting both global and local RNA interactions.The transient formation of an intermolecular kissing complex is required for RNA dimerization during the life cycle of retroviruses (11-15), and for the formation of some antisensetarget complexes (16,17). Kissing complexes in which the loop nucleotides are complementary can form stable dimers that contain intermolecular base pairs between the loop nucleotides only. Other stem-loops with more extensive complementarity sometimes form unstable kissing complexes, which are quickly remodeled into stable duplex or cruciform isoforms (18)(19)(20). Secondary structure remodeling involves breaking intramolecular base pairs in each hairpin and using the bases to form intermolecular helices.The VS ribozyme contains a kissing interaction that is required for self-cleavage of the wild-type RNA in vitro (6). This interaction involves Watson-Crick bas...

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Section: Chemical Modification Reveals the Kissing Interaction Is Reqsupporting

confidence: 90%

Intramolecular secondary structure rearrangement by the kissing interaction of the Neurospora VS ribozyme

Andersen

Collins

2001

Proc. Natl. Acad. Sci. U.S.A.

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“…Clearly, A756 must be a candidate for this role in the VS ribozyme. The rate of the cleavage reaction is not significantly dependent on pH (Rastogi and Collins 1998), unlike that for the hammerhead reaction (Dahm et al 1993), but pH dependence corresponding to a pK a of 5.6 has been observed for the ligation reaction (McLeod and Lilley 2004). The restoration of good ligation activity in VS ribozyme variants with nucleotide bases of reduced pK a at lowered pH and stimulation of activity in variants with nucleotide bases of increased pK a point to a requirement for a protonated base at position 756 (Jones and Strobel 2003).…”

Section: Possible Catalytic Mechanismsmentioning

confidence: 92%

The Varkud satellite ribozyme

Lilley

2004

RNA

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The VS ribozyme is the largest nucleolytic ribozyme, for which there is no crystal structure to date. The ribozyme consists of five helical sections, organized by two three-way junctions. The global structure has been determined by solution methods, particularly FRET. The substrate stem-loop binds into a cleft formed between two helices, while making a loop-loop contact with another section of the ribozyme. The scissile phosphate makes a close contact with an internal loop (the A730 loop), the probable active site of the ribozyme. This loop contains a particularly critical nucleotide A756. Most changes to this nucleotide lead to three-orders of magnitude slower cleavage, and the Watson-Crick edge is especially important. NAIM experiments indicate that a protonated base is required at this position for the ligation reaction. A756 is thus a strong candidate for nucleobase participation in the catalytic chemistry.

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“…Double-stranded PCR fragments coding for the J236-VS, J236-A 6 -bp-VS, and J236-ΔA 6 -VS RNAs and flanked by a T7 promoter, were inserted into the HindIII/EcoRI sites of the pTZ19R-derived pTR-4 vector (Rastogi and Collins 1998) to generate the pJ236, pJ236-ΔA 6 , and pJ236-A 6 -bp plasmids. These plasmids were fully linearized using EcoRI (New England Biolabs) and used for transcription of the J236, J236-ΔA 6 , and J236-A 6 -bp RNAs (Fig.…”

Section: Plasmidsmentioning

confidence: 99%

The NMR structure of the II–III–VI three-way junction from the Neurospora VS ribozyme reveals a critical tertiary interaction and provides new insights into the global ribozyme structure

Bonneau

Girard

Lemieux

et al. 2015

RNA

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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 threedimensional architecture of this unique ribozyme.

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Smaller, faster ribozymes reveal the catalytic core of Neurospora VS RNA

Cited by 46 publications

References 29 publications

Intramolecular secondary structure rearrangement by the kissing interaction of the Neurospora VS ribozyme

Intramolecular secondary structure rearrangement by the kissing interaction of the Neurospora VS ribozyme

The Varkud satellite ribozyme

The NMR structure of the II–III–VI three-way junction from the Neurospora VS ribozyme reveals a critical tertiary interaction and provides new insights into the global ribozyme structure

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