Published: Vol 6, Iss 17, Sep 5, 2016 DOI: 10.21769/BioProtoc.1917 Views: 9291
Reviewed by: Arsalan DaudiLongping Victor TseAnonymous reviewer(s)
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Abstract
This protocol describes the expression, purification and crystallization of a ternary protein-protein-RNA complex, consisting of the two RNA recognition motifs (RRMs) of Sex-lethal (Sxl), the first of five cold shock domains of Upstream-of-N-Ras (Unr), and an 18-nucleotide region of msl2 mRNA, called the F fragment (Hennig et al., 2014).The biological role of the complex is the translational repression of msl2 mRNA, preventing the formation of the dosage compensation complex and subsequent 2-fold hypertranscription of X-linked genes in Drosophila females. As orthologous RRM-containing proteins and Unr exist in humans, similar complexes potentially also form during translational repression in vertebrates. The protocol describes the in vitro assembly of the complex and its purification followed by crystallization for X-ray crystallography structure determination. Part of the protocol has been published elsewhere (Hennig et al., 2013 and 2014), but some parts are described here in more detail.
Materials and Reagents
Equipment
Procedure
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Acknowledgments
We thank the crystallization facility at the Max-Planck-Institute for Biochemistry, Martinsried for initial crystallization screens and Arie Geerlof for protein expressions and purifications. J.H. acknowledges postdoctoral fellowships from the Swedish Research Council (Vetenskapsradet) and the European Molecular Biology Organization (EMBO, ALTF276-2010). M.S. acknowledges the Deutsche Forschungsgemeinschaft (DFG), grants SFB1035 and GRK1721.
References
Article Information
Copyright
© 2016 The Authors; exclusive licensee Bio-protocol LLC.
How to cite
Hennig, J. and Sattler, M. (2016). Protein Expression, Purification and Crystallization of the Sxl-Unr-msl2 Ribonucleoprotein Complex. Bio-protocol 6(17): e1917. DOI: 10.21769/BioProtoc.1917.
Category
Biochemistry > Protein > Structure
Biochemistry > RNA > RNA-protein interaction
Biochemistry > Protein > Isolation and purification
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