Small self-cleaving nucleolytic ribozymes contain catalytic domains that accelerate site-specific cleavage/ligation of phosphodiester backbones. We report on the 2.9-Å crystal structure of the env22 twister ribozyme, which adopts a compact tertiary fold stabilized by co-helical stacking, double-pseudoknot formation and long-range pairing interactions. The U-A cleavage site adopts a splayed-apart conformation with the modeled 2′-O of U positioned for in-line attack on the adjacent to-be-cleaved P-O5′ bond. Both an invariant guanosine and a Mg2+ are directly coordinated to the non-bridging phosphate oxygens at the U-A cleavage step, with the former positioned to contribute to catalysis and the latter to structural integrity. The impact of key mutations on cleavage activity identified an invariant guanosine that contributes to catalysis. Our structure of the in-line aligned env22 twister ribozyme is compared with two recently-reported twister ribozymes structures, which adopt similar global folds, but differ in conformational features around the cleavage site.

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 pKa value of 5.1 was determined for a 13C2-labeled adenine at this position in the twister ribozyme, which is significantly shifted compared to the pKa 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 Mg2+ 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 Mn2+ or Cd2+ 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′-OCH3-U5 modified twister ribozyme.

The chemical synthesis of ribonucleic acids (RNA) with novel chemical modifications is largely driven by the motivation to identify eligible functional probes for the various applications in life sciences. To this end, we have a strong focus on the development of novel fluorinated RNA derivatives that are powerful in NMR spectroscopic analysis of RNA folding and RNA ligand interactions. Here, we report on the synthesis of 2′-SCF3 pyrimidine nucleoside containing oligoribonucleotides and the comprehensive investigation of their structure and base pairing properties. While this modification has a modest impact on thermodynamic stability when it resides in single-stranded regions, it was found to be destabilizing to a surprisingly high extent when located in double helical regions. Our NMR spectroscopic investigations on short single-stranded RNA revealed a strong preference for C2′-endo conformation of the 2′-SCF3 ribose unit. Together with a recent computational study (L. Li, J. W. Szostak, J. Am. Chem. Soc. 2014, 136, 2858–2865) that estimated the extent of destabilization caused by a single C2′-endo nucleotide within a native RNA duplex to amount to 6 kcal mol−1 because of disruption of the planar base pair structure, these findings support the notion that the intrinsic preference for C2′-endo conformation of 2′-SCF3 nucleosides is most likely responsible for the pronounced destabilization of double helices. Importantly, we were able to crystallize 2′-SCF3 modified RNAs and solved their X-ray structures at atomic resolution. Interestingly, the 2′-SCF3 containing nucleosides that were engaged in distinct mismatch arrangements, but also in a standard Watson–Crick base pair, adopted the same C3′-endo ribose conformations as observed in the structure of the unmodified RNA. Likely, strong crystal packing interactions account for this observation. In all structures, the fluorine atoms made surprisingly close contacts to the oxygen atoms of the corresponding pyrimidine nucleobase (O2), and the 2′-SCF3 moieties participated in defined water-bridged hydrogen-bonding networks in the minor groove. All these features allow a rationalization of the structural determinants of the 2′-SCF3 nucleoside modification and correlate them to base pairing properties.

Molecular sieves ensure proper pairing of tRNAs and amino acids during aminoacyl-tRNA biosynthesis, thereby avoiding detrimental effects of mistranslation on cell growth and viability. Mischarging errors are often corrected through the activity of specialized editing domains present in some aminoacyl-tRNA synthetases or via single-domain -editing proteins. ProXp-ala is a ubiquitous-editing enzyme that edits Ala-tRNA, the product of Ala mischarging by prolyl-tRNA synthetase, although the structural basis for discrimination between correctly charged Pro-tRNA and mischarged Ala-tRNA is unclear. Deacylation assays using substrate analogs reveal that size discrimination is only one component of selectivity. We used NMR spectroscopy and sequence conservation to guide extensive site-directed mutagenesis of ProXp-ala, along with binding and deacylation assays to map specificity determinants. Chemical shift perturbations induced by an uncharged tRNA acceptor stem mimic, microhelix, or a nonhydrolyzable mischarged Ala-microhelix substrate analog identified residues important for binding and deacylation. Backbone N NMR relaxation experiments revealed dynamics for a helix flanking the substrate binding site in free ProXp-ala, likely reflecting sampling of open and closed conformations. Dynamics persist on binding to the uncharged microhelix, but are attenuated when the stably mischarged analog is bound. Computational docking and molecular dynamics simulations provide structural context for these findings and predict a role for the substrate primary α-amine group in substrate recognition. Overall, our results illuminate strategies used by a-editing domain to ensure acceptance of only mischarged Ala-tRNA, including conformational selection by a dynamic helix, size-based exclusion, and optimal positioning of substrate chemical groups.

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...