Chapter Six - Strategies for crystallization of natural ribozymes - Archive ouverte HAL
Chapitre D'ouvrage Année : 2020

Chapter Six - Strategies for crystallization of natural ribozymes

Résumé

The group of ribozymes acting like turnover enzymes is up to now restricted to the ribonuclease P [1], the ribosome [2] and the spliceosome [3]. Other natural ribozymes are endonucleolytic RNAs, catalyzing the reversible cleavage of a phosphodiester bond within their own chain. This group contains the hammerhead [4], hairpin [5], hepatitis delta virus (HdV) [6], and the Varkud satelitte (VS) [7] ribozymes, and also group I [8, 9] and group II introns [9]. More recently discovered ribozymes include twister [10], twister sister, pistol and hatchet [11], as well as GlmS [12], which is also a riboswitch. When the cleavage intervenes at the boundaries of the ribozyme, like in the case of the HdV ribozyme, the structure of the initial, or pre-catalytic RNA is expected to be quite similar to the 3' cleavage product. Yet, most frequently, cleavage intervenes at an internal position. In the course of the reaction, the amount of pre-cleavage form decreases in solution until the catalytic equilibrium is reached. As fragments of the ribozyme itself, the RNA cleavage products may not be able to fold properly since cleavage affects the higher order structure of the RNA by splitting structural domains. Therefore, a solution of active ribozymes ends up as a mix of different characteristic RNA precursors and products, to which should be added shorter species resulting from the degradation of unstable cleavage products. Crystallization of biomolecules requires chemical and conformational homogeneity [13]. RNA incubation in crystallization droplets usually lasts a couple of days or weeks at temperatures ranging from 15°C to 37°C, at pH comprised between 6 to 8, in the presence of monovalent and divalent and/or trivalent ions, like magnesium salts or
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Dates et versions

hal-03002014 , version 1 (12-11-2020)

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Benoit Masquida, D Sibrikova, Maria Costa. Chapter Six - Strategies for crystallization of natural ribozymes. Ribozymes, Wiley-VCH, 2020, ⟨10.1002/9783527814527.ch30⟩. ⟨hal-03002014⟩
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