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Design of cross-linked RNA/protein complexes for structural studies

Author:
Dégut, Clément, Schwarz, Veronika, Ponchon, Luc, Barraud, Pierre, Micura, Ronald, Tisné, Carine
Source:
Biochimie 2019
ISSN:
0300-9084
Subject:
Thermus thermophilus, crosslinking, crystallization, electrostatic interactions, enzymes, transfer RNA
Abstract:
Crystallographic studies of RNA/protein complexes are primordial for the understanding of recognition determinants and catalytic mechanisms in the case of enzymes. However, due to the flexibility and propensity to conformational heterogeneity of RNAs, as well as the mostly electrostatic interactions of RNA/protein complexes, they are difficult to crystallize. We present here a method to trap the two interacting partners in a covalent complex, based on a modified reactive RNA allowing the use of the full range of common crystallogenesis tools. We demonstrate the practicability of our approach with the production of a covalent complex of the Thermus thermophilus m1A58 tRNA modification enzyme, and a modified stem loop mimicking the natural substrate of the enzyme.
Agid:
6358712