A Mini‐Twister Variant and Impact of Residues/Cations on the Phosphodiester Cleavage of this Ribozyme Class

Abstract

Nucleolytic ribozymes catalyze site-specific cleavage of their phosphodiester backbones. A minimal version of the twister ribozyme is reported that lacks the phylogenetically conserved stem P1 while retaining wild-type activity. Atomic mutagenesis revealed that nitrogen atoms N1 and N3 of the adenine-6 at the cleavage site are indispensable for cleavage. By NMR spectroscopy, a pK a value of 5.1 was determined for a 13 C2-labeled adenine at this position in the twister ribozyme, which is significantly shifted compared to the pK a of the same adenine in the substrate alone. This finding pinpoints at a potential role for adenine-6 in the catalytic mechanism besides the previously identified invariant guanine-48 and a Mg 2+ ion, both of which are directly coordinated to the non-bridging oxygen atoms of the scissile phosphate; for the latter, additional evidence stems from the observation that Mn 2+ or Cd 2+ accelerated cleavage of phosphorothioate substrates. The relevance of this metal ion binding site is further emphasized by a new 2.6 Å X-ray structure of a 2′-OCH 3 -U5 modified twister ribozyme. Keywordsmetal ion rescue; nucleoside modifications; oligoribonucleotides; perturbed pK a ; solid-phase synthesis Small self-cleaving ribozymes are widely distributed in nature [1] and are essential for rolling-circle-based replication of satellite RNAs. [2,3] Among them, the hepatitis delta virus (HDV) ribozyme [4][5][6][7][8] employs a divalent cation in the active site for catalysis, while the remaining small self-cleaving ribozymes including hammerhead, [2,9,10] hairpin, [3,[11][12][13] glmS, [14][15][16] and Varkud Satellite [17] employ principles of general acid-base and electrostatics for catalysis. Very recently, a new class of nucleolytic ribozymes (termed twister) has been discovered, [18] and soon thereafter, crystal structures were published that revealed a common double-pseudoknot overall architecture for the twister ribozyme but showed clear distinctions in residue and divalent cation alignments at the cleavage site. [19][20][21] While the O. sativa twister ribozyme was off-line orthogonally aligned with a fully base-paired stem P1, [19,20] the env22 twister ribozyme was in-line oriented at the cleavage step A6-U5, with a Mg 2+ coordinated to the scissile phosphate. Furthermore, for the env22 twister ribozyme, stem P1 formed only the two central base pairs (Figure 1) while the neighboring nucleotides U1 and U4 were instead engaged in stacked base triplet interactions (U4-A49-A34 and U1-A50-U33; Figure 1B). [21] Košutić et al.Page 2 Angew Chem Int Ed Engl. Author manuscript; available in PMC 2016 January 16. Author Manuscript Author ManuscriptAuthor Manuscript Author ManuscriptThese contrasting observations were the starting point for the present investigation. A thorough comparison of the two structures (PDB: 4OJI for O. sativa and PDB: 4RGF for env22) revealed that the conserved adenosine (A6; env22 numbering is used throughout) at the cleavage site adopts nearly identical conformations involving ex...

An Ontology for Facilitating Discussion of Catalytic Strategies of RNA-Cleaving Enzymes

“…Specific example (Twr): A1(2AP) mutation 39 (k/k' >10 5 ) that leads to disruption of a critical anchoring interaction between A1:N2 and the NPOs of C16 and C17. Note (example of functional blurring): these interactions also play a role in secondary δ catalysis by shifting the pK a of A1:N3 toward neutrality in the ribozyme environment.…”

Aminoglycoside antibiotics can inhibit or activate twister ribozyme cleavage

“…Each of the crystal structures emphasizes an important role for the highly conserved guanosine that coordinates directly to the nonbridging phosphate oxygen in the catalysis of the U‐A phosphate cleavage [2–5,38]. To investigate the effects of aminoglycoside binding on the structure of the ribozyme RNA, we used selective 2′‐hydroxyl acylation analysed by primer extension (SHAPE) of twister RNA upon addition of paromomycin or sisomicin or in their absence.…”

“…The ribozyme constructs for SHAPE analysis were prepared by splinted enzymatic ligation of two chemically synthesized RNA fragments using T4 DNA ligase (NEB, Ipswich, MA, USA), as previously published [35,36,38]. Briefly, 20 µ m of each RNA strand, 20 µ m of a DNA splint oligonucleotide and a final ligase concentration of 0.5 U·µL −1 in a total volume of 50 µL were incubated for 3 h at 37 °C.…”

Faculty Opinions recommendation of Cleaning up mechanistic debris generated by twister ribozymes using computational RNA enzymology.

“…The recent discovery of the twister ribozyme from comparative genomics 8 sparked the generation of a wealth of structural data from X-ray crystallography 31,[43][44][45] that has been discussed in a recent review. 46 The twister ribozyme secondary structure (Figure 1a) consists of three alternating stems (P1, P2, and P4) and loops (L1, L2, and L4) which are organized into a compact fold (Figure 1b) by the tertiary contacts formed by two pseudoknots T1 and T2.…”