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Mg2+ Binding Promotes SLV as a Scaffold in Varkud Satellite Ribozyme SLI-SLV Kissing Loop Junction.

ABSTRACT: 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.

SUBMITTER: Bergonzo C

PROVIDER: S-EPMC5529310 | biostudies-literature | 2017 Jul

REPOSITORIES: biostudies-literature

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Mg2+ Binding Promotes SLV as a Scaffold in Varkud Satellite Ribozyme SLI-SLV Kissing Loop Junction.

Bergonzo Christina C Cheatham Thomas E TE

Biophysical journal 20170629 2

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 loo ...[more]

PMID: 28669407

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Structural insights into substrate recognition by the Neurospora Varkud satellite ribozyme: importance of U-turns at the kissing-loop junction.

Project description:Substrate recognition by the Neurospora Varkud satellite ribozyme depends on the formation of a magnesium-dependent kissing-loop interaction between the stem-loop I (SLI) substrate and stem-loop V (SLV) of the catalytic domain. From mutagenesis studies, it has been established that this I/V kissing-loop interaction involves three Watson-Crick base pairs and is associated with a structural rearrangement of the SLI substrate that facilitates catalysis. Here, we report the NMR structural characterization of this I/V kissing-loop using isolated stem-loops. NMR studies were performed on different SLI/SLV complexes containing a common SLV and shiftable, preshifted, or double-stranded SLI variants. These studies confirm the presence of three Watson-Crick base pairs at the kissing-loop junction and provide evidence for the structural rearrangement of shiftable SLI variants upon SLV binding. NMR structure determination of an SLI/SLV complex demonstrates that both the SLI and SLV loops adopt U-turn structures, which facilitates intermolecular Watson-Crick base pairing. Several other interactions at the I/V interface, including base triples and base stacking, help create a continuously stacked structure. These NMR studies provide a structural basis to understand the stability of the I/V kissing-loop interaction and lead us to propose a kinetic model for substrate activation in the VS ribozyme.

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A remarkably stable kissing-loop interaction defines substrate recognition by the Neurospora Varkud Satellite ribozyme.

Project description:Kissing loops are tertiary structure elements that often play key roles in functional RNAs. In the Neurospora VS ribozyme, a kissing-loop interaction between the stem-loop I (SLI) substrate and stem-loop V (SLV) of the catalytic domain is known to play an important role in substrate recognition. In addition, this I/V kissing-loop interaction is associated with a helix shift in SLI that activates the substrate for catalysis. To better understand the role of this kissing-loop interaction in substrate recognition and activation by the VS ribozyme, we performed a thermodynamic characterization by isothermal titration calorimetry using isolated SLI and SLV stem-loops. We demonstrate that preshifted SLI variants have higher affinity for SLV than shiftable SLI variants, with an energetic cost of 1.8-3 kcal/mol for the helix shift in SLI. The affinity of the preshifted SLI for SLV is remarkably high, the interaction being more stable by 7-8 kcal/mol than predicted for a comparable duplex containing three Watson-Crick base pairs. The structural basis of this remarkable stability is discussed in light of previous NMR studies. Comparative thermodynamic studies reveal that kissing-loop complexes containing 6-7 Watson-Crick base pairs are as stable as predicted from comparable RNA duplexes; however, those with 2-3 Watson-Crick base pairs are more stable than predicted. Interestingly, the stability of SLI/ribozyme complexes is similar to that of SLI/SLV complexes. Thus, the I/V kissing loop interaction represents the predominant energetic contribution to substrate recognition by the trans-cleaving VS ribozyme.

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Crystal structure of the Varkud satellite ribozyme.

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Nucleobase-mediated general acid-base catalysis in the Varkud satellite ribozyme.

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NMR structure of the active conformation of the Varkud satellite ribozyme cleavage site.

Project description: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 similarities, but also significant differences, with the previously determined structures of the inactive internal loop. The active internal loop displays different base-pairing interactions and forms a novel RNA fold composed exclusively of sheared G-A base pairs. From chemical-shift mapping we identified two Mg2+ binding sites in the active internal loop. One of the Mg2+ binding sites forms in the active but not the inactive conformation of the internal loop and is likely important for catalysis. Using the structure comparison program mc-search, we identified the active internal loop fold in other RNA structures. In Thermus thermophilus 16S rRNA, this RNA fold is directly involved in a long-range tertiary interaction. An analogous tertiary interaction may form between the active internal loop of the substrate and the catalytic domain of the VS ribozyme. The combination of NMR and bioinformatic approaches presented here has identified a novel RNA fold and provides insights into the structural basis of catalytic function in the Neurospora VS ribozyme.

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Project description:The Neurospora VS ribozyme is a catalytic RNA that has the unique ability to specifically recognize and cleave a stem-loop substrate through formation of a highly stable kissing-loop interaction (KLI). In order to explore the engineering potential of the VS ribozyme to cleave alternate substrates, we substituted the wild-type KLI by other known KLIs using an innovative engineering method that combines rational and combinatorial approaches. A bioinformatic search of the protein data bank was initially performed to identify KLIs that are structurally similar to the one found in the VS ribozyme. Next, substrate/ribozyme (S/R) pairs that incorporate these alternative KLIs were kinetically and structurally characterized. Interestingly, several of the resulting S/R pairs allowed substrate cleavage with substantial catalytic efficiency, although with reduced activity compared to the reference S/R pair. Overall, this study describes an innovative approach for RNA engineering and establishes that the KLI of the trans VS ribozyme can be adapted to cleave other folded RNA substrates.

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