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Unwinding the twister ribozyme: from structure to mechanism

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The twister ribozyme motif has been identified by bioinformatic means very recently. Currently, four crystal structures with ordered active sites together with a series of chemical and biochemical data provide insights into how this RNA accomplishes its efficient self‐cleavage. Of particular interest for a mechanistic proposal are structural distinctions observed in the active sites that concern the conformation of the U‐A cleavage site dinucleotide (in‐line alignment of the attacking 2′‐O nucleophile to the to‐be‐cleaved PO5′ bond versus suboptimal alignments) as well as the presence/absence of Mg2+ ions at the scissile phosphate. All structures support the notion that an active site guanine and the conserved adenine at the cleavage site are important contributors to cleavage chemistry, likely being involved in general acid base catalysis. Evidence for innersphere coordination of a Mg2+ ion to the pro‐S nonbridging oxygen of the scissile phosphate stems from two of the four crystal structures. Together with the finding of thio/rescue effects for phosphorothioate substrates, this suggests the participation of divalent ions in the overall catalytic strategy employed by twister ribozymes. In this context, it is notable that twister retains wild‐type activity when the phylogenetically conserved stem P1 is deleted, able to cleave a single nucleotide only. WIREs RNA 2017, 8:e1402. doi: 10.1002/wrna.1402

Comparison of env22 (5DUN and 4RGE), and O. sativa (4OJI) twister ribozymes—overall folds. (a) Leontis–Westhof presentation (left) and three‐dimensional structure in cartoon presentation (right) of the dU modified env22 ribozyme, (b) the 2′‐OCH3U modified env22 ribozyme, and (c) the dU modified O. sativa ribozyme. Note the differences in segment P1 (green): ‘back‐folding’ of nucleosides (U1 and U4) of segment P1 for env22 to form base triplets (also see panel (a)), and fully Watson–Crick base‐paired stem P1 for O. sativa, respectively. Black triangles indicate the cleavage positions.
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Twister ribozymes. (a) Consensus sequence and secondary structure model for the twister RNA motif (adapted from Refs ); Watson–Crick base‐paired stems and pseudoknots are defined by P# and T#, respectively; terminal and internal loops are defined by L#. Detailed sequences of the O. sativa (b) and the env22 (c) twister ribozyme constructs used for structural and biochemical studies. For the env9 twister sequence and secondary structure, see Ref .
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RNA phosphodiester cleavage by phosphoester transfer involving the 2′‐hydroxyl group. The internucleotide linkage (‘scissile’ phosphate) passes through a pentacoordinate transition state that results in two cleavage products carrying either a 2′,3′‐cyclic phosphate terminus or a 5′‐hydroxyl terminus. The four catalytic strategies that can impact on the reaction are: α, in‐line nucleophilic attack, SN2‐type (blue); β, neutralization of the (developing) negative charge on nonbridging phosphate oxygens (purple); γ, deprotonation of the 2′‐hydroxyl group (red); and δ, neutralization of negative charge on the 5′‐oxygen atom by protonation (green).
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Comparison to pistol and hammerhead ribozymes—active sites and the to‐be‐cleaved dinucleotide units (the latter highlighted in yellow). (a) dG–U arrangement in the 2.7 Å resolution structure of the env25 pistol ribozyme (PDB: 5K7V) with emphasis on the position of the C2′ of dG relative to the PO5′ bond (for a presentation with modeled 2′‐OH on C2′, see Ref ). (b) dC–U arrangement in the 3.0 Å resolution structure of the RzB hammerhead ribozyme (PDB: 5DI2). Directions for in‐line attack of the O2′ nucleophile at the to‐be‐cleaved PO5′ bond are indicated by cyan arrows. C2′ positions (where the 2′‐OH nucleophiles are attached in the corresponding functional RNAs) are indicated by black arrows; τ describes the angle O2′ (of nucleotide N − 1) to P–O5′ (of nucleotide N + 1) according to reference : in‐line alignment implies a τ of 180°.
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Comparison of env22 (5DUN and 4RGE), O. sativa (4OJI) and env9 (4QJH) twister ribozymes—active sites and the to‐be‐cleaved dinucleotide units (the latter highlighted in yellow). (a) dU–A arrangement in the 2.9 Å resolution structure of the env22 twister ribozyme with emphasis on the position of the C2′ of dU relative to the PO5′ bond (for a presentation with modeled 2′‐OH on C2′, see Ref ). (b) dU–A arrangement in the 2.3 Å resolution structure of the O. sativa twister ribozyme (PDB: 4OJI); local conformational heterogeneity at the scissile phosphate was observed; fitting this to a double conformation (as shown) improved the electron density map (for F oF c maps, see Figure S3 of Ref ). (c) 2′‐OCH3U–A arrangement in the 2.6 Å resolution structure of the env22 twister ribozyme (PDB: 5DUN). (d) dU–A arrangement in the 4.1 Å resolution structure of the env9 twister ribozyme (PDB: 4QJH). Directions for in‐line attack of the O2′ nucleophile at the to‐be‐cleaved PO5′ bond are indicated by cyan arrows. C2′ positions (where the 2′‐OH nucleophiles are attached in the corresponding functional RNAs) are indicated by black arrows; τ describes the angle O2′ (of nucleotide N − 1) to P–O5′ (of nucleotide N + 1) according to Ref : in‐line alignment implies a τ of 180°.
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Single nucleotide cleavage of the twister ribozyme. (a) The twister ribozyme does not require formation of the phylogenetically conserved stem P1 for efficient cleavage. Exemplary HPLC cleavage assay for a 5′‐truncated substrate RNA, showing that a single nucleotide (U5) is cleaved. Conditions: 2 mm MgCl2, 100 mm KCl, 30 mm HEPES, pH 7.5, 23°C. (b) Cleavage kinetics of a twister ribozyme lacking stem P1 (‘mini‐twister’) analyzed by ensemble 2‐aminopurine (Ap) fluorescence spectroscopy. The nonconserved U5 was replaced by Ap (in red letters). Note that Ap fluorescence decreases during the course of cleavage; this observation hints at the very exposed and unshielded arrangement that the nucleobase has to adopt (active conformation) to become cleaved; conditions: cRNA = 0.3 μM, 50 mM potassium 3‐(N‐morpholino)propanesulfonate (KMOPS), 100 mM KCl, 15°C, pH 7.5; mixing was performed manually in less than 2 seconds resulting in 10 mM Mg2+ concentration (see also Figure S2 in Ref ).
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Mechanistic proposal for phosphodiester cleavage in the twister ribozyme, exemplified for the env22 RNA. (a) Guanine‐48 can stabilize the transition state and may also be involved in activation of the U5 2′‐OH. The Mg2+ ion coordinated to the scissile phosphate (and additionally clamped to the successive phosphate unit) also stabilizes the transition state and may assist to neutralize the developing charge on the leaving O5′ through donation of a proton via a coordinated water molecule. (b) A further candidate for acting as general acid is adenine‐6. This adenine possesses a shifted pK a of 5.1 as determined by NMR spectroscopy, and may donate its proton to the leaving O5′. From a structural perspective, however, only N3 is appropriately positioned in vicinity to O5′ (top), and not N1 (bottom inset); the latter representing the preferred protonation site of a protonated adenine (>96% versus 0.7% for N7/N3 according to reference ). Additional studies are required to complete our understanding of the mechanism for twister ribozymes that most likely use a combination of catalytic strategies as discussed in the main text.
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RNA Structure and Dynamics > RNA Structure, Dynamics and Chemistry
RNA-Based Catalysis > RNA-Mediated Cleavage
Regulatory RNAs/RNAi/Riboswitches > Riboswitches

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