In-line alignment and Mg2+ coordination at the cleavage site of the env22 twister ribozyme

Ren, Aiming; Košutić, Marija; Rajashankar, Kanagalaghatta R.; Frener, Marina; Santner, Tobias; Westhof, Éric; Micura, Ronald; Patel, Dinshaw J.

doi:10.1038/ncomms6534

Cited by 85 publications

(240 citation statements)

References 36 publications

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“…One caveat is that RA does not correlate linearly with the intrinsic activity (that is, k cat ) of the ribozyme because the speed of the wild‐type ribozyme reported under similar conditions ( k cat =1–10 min −1 , corresponding to a half‐life of 1 min or less)6, 7b is much faster than the timescale of our experiment (120 min). Therefore, a moderate reduction in RA actually corresponds to a more drastic decrease in the rate constant.…”

mentioning

confidence: 57%

High‐Throughput Mutational Analysis of a Twister Ribozyme

Kobori

Yokobayashi

2016

Angew Chem Int Ed

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Recent discoveries of new classes of self‐cleaving ribozymes in diverse organisms have triggered renewed interest in the chemistry and biology of ribozymes. Functional analysis and engineering of ribozymes often involve performing biochemical assays on multiple ribozyme mutants. However, because each ribozyme mutant must be individually prepared and assayed, the number and variety of mutants that can be studied are severely limited. All of the single and double mutants of a twister ribozyme (a total of 10 296 mutants) were generated and assayed for their self‐cleaving activity by exploiting deep sequencing to count the numbers of cleaved and uncleaved sequences for every mutant. Interestingly, we found that the ribozyme is highly robust against mutations such that 71 % and 30 % of all single and double mutants, respectively, retain detectable activity under the assay conditions. It was also observed that the structural elements that comprise the ribozyme exhibit distinct sensitivity to mutations.

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confidence: 57%

High‐Throughput Mutational Analysis of a Twister Ribozyme

Kobori

Yokobayashi

2016

Angew Chem Int Ed

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“…From the analysis of the genoma, twister RNAs have been suggested to be similar to hammerhead in size and structural complexity, and their self-cleavage rate constants are also similar. Nevertheless, its overall structure is distinct from all the other known small ribozymes, as recently confirmed by X-ray diffraction studies [17][18][19][20]. As stated by Patel, Kreutz, Micura and co-workers [20], the analysis of these crystal structures reveals a common double-pseudoknot overall architecture but with noticeable differences in residues and divalent cation alignments in the active sites.…”

mentioning

confidence: 62%

Molecular mechanism of the site-specific self-cleavage of the RNA phosphodiester backbone by a twister ribozyme

et al. 2017

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isotope effects on the nucleophile and leaving oxygen atoms, in very good agreement with experiments, also support this description. Nevertheless, the free energy profiles in the enzyme and in solution are almost coincident which, despite that the rate-limiting activation free energy is in very good agreement with experimental data of counterpart reactions in solution, rule out this substrate-assisted catalysis mechanism for the twister ribozyme from O. sativa. Catalysis must come from the role of alternative acid-base species not available in aqueous solution, but the rate-limiting transition state must be associated with the P-O5′ bond cleavage.

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“…Therefore, the large dependency of hatchet ribozymes on Mg 2+ also could be purely structural, rather than an Biochemical analysis of hatchet ribozymes www.rnajournal.org 1847 indication of direct participation of metal ions in catalysis. However, another structural model places a Mg 2+ ion in coordination with the cleavage site phosphate moiety (Ren et al 2014), but this metal ion is absent in an additional structural model of the twister ribozyme active site (Eiler et al 2014). Given these uncertainties, the precise roles played by divalent cations cannot yet be established for twister ribozymes despite considerable biochemical and structural data.…”

Section: The Effects Of Various Mono-and Divalent Metal Ions On Hatchmentioning

confidence: 99%

Biochemical analysis of hatchet self-cleaving ribozymes

Lünse

Harris

et al. 2015

RNA

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Hatchet RNAs are members of a novel self-cleaving ribozyme class that was recently discovered by using a bioinformatics search strategy. The consensus sequence and secondary structure of this class includes 13 highly conserved and numerous other modestly conserved nucleotides interspersed among bulges linking four base-paired substructures. A representative hatchet ribozyme from a metagenomic source requires divalent ions such as Mg 2+ to promote RNA strand scission with a maximum rate constant of ∼4 min −1 . As with all other small self-cleaving ribozymes discovered to date, hatchet ribozymes employ a general mechanism for catalysis involving the nucleophilic attack of a ribose 2 ′ -oxygen atom on an adjacent phosphorus center. Kinetic characteristics of the reaction demonstrate that members of this ribozyme class have an essential requirement for divalent metal ions and that they might have a complex active site that employs multiple catalytic strategies to accelerate RNA cleavage by internal phosphoester transfer.

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In-line alignment and Mg2+ coordination at the cleavage site of the env22 twister ribozyme

Cited by 85 publications

References 36 publications

High‐Throughput Mutational Analysis of a Twister Ribozyme

High‐Throughput Mutational Analysis of a Twister Ribozyme

Molecular mechanism of the site-specific self-cleavage of the RNA phosphodiester backbone by a twister ribozyme

Biochemical analysis of hatchet self-cleaving ribozymes

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