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

Clément Dégut; Veronika Schwarz; Luc Ponchon; Pierre Barraud; Ronald Micura; Carine Tisné

doi:10.1016/j.biochi.2019.03.021

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

Biochimie. 2019 Sep:164:95-98. doi: 10.1016/j.biochi.2019.03.021. Epub 2019 Mar 30.

Authors

Clément Dégut¹, Veronika Schwarz², Luc Ponchon¹, Pierre Barraud³, Ronald Micura², Carine Tisné⁴

Affiliations

¹ Laboratoire de Cristallographie et RMN Biologiques, CNRS, Université Paris Descartes, Sorbonne Paris Cité, Paris, France.
² Institute of Organic Chemistry and Center for Molecular Biosciences Innsbruck CMBI, Leopold-Franzens University, Innsbruck, Austria.
³ Laboratoire de Cristallographie et RMN Biologiques, CNRS, Université Paris Descartes, Sorbonne Paris Cité, Paris, France; Laboratoire d'Expression Génétique Microbienne, CNRS, Université Paris Diderot, Sorbonne Paris Cité, Institut de Biologie Physico-Chimique, IBPC, Paris, France.
⁴ Laboratoire de Cristallographie et RMN Biologiques, CNRS, Université Paris Descartes, Sorbonne Paris Cité, Paris, France; Laboratoire d'Expression Génétique Microbienne, CNRS, Université Paris Diderot, Sorbonne Paris Cité, Institut de Biologie Physico-Chimique, IBPC, Paris, France. Electronic address: carine.tisne@cnrs.fr.

PMID: 30940603
DOI: 10.1016/j.biochi.2019.03.021

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.

Keywords: Covalent trapping; Cross-link; Methyltransferase; RNA-Protein complex; TrmI; tRNA.

MeSH terms

Bacterial Proteins / chemistry
Crystallography
Models, Molecular
Protein Binding
RNA, Bacterial / chemistry
RNA, Transfer / chemistry*
Substrate Specificity
Thermus thermophilus / enzymology
Thermus thermophilus / genetics
tRNA Methyltransferases / chemistry*

Substances

Bacterial Proteins
RNA, Bacterial
RNA, Transfer
tRNA Methyltransferases