期刊
BIOCHEMICAL JOURNAL
卷 449, 期 -, 页码 719-728出版社
PORTLAND PRESS LTD
DOI: 10.1042/BJ20121211
关键词
affinity purification; cryo-electron microscopy (cryo-EM); mRNA DNA chimaera; ribosome; translation initiation
资金
- CNRS (Centre National de la Recherche Scientifique)
- Association pour la Recherche sur le Cancer [3814]
- Agence Nationale pour la Recherche [ANR-06-BLAN-0206-01, ANR-2011-SVSE8-025-01]
- Agence Nationale de la Recherche (ANR) [ANR-06-BLAN-0206] Funding Source: Agence Nationale de la Recherche (ANR)
Detailed knowledge of the structure of the ribosomal particles during their assembly on mRNA is a prerequisite for understanding the intricate translation initiation process. In vitro preparation of eukaryotic translation initiation complexes is limited by the rather tricky. assembly from individually purified ribosomal subunits, initiation factors and initiator tRNA. In order to directly isolate functional complexes from living cells, methods based on affinity tags have been developed which, however, often suffer from non-specific binding of proteins and/or RNAs. In the present study we present a novel method designed for the purification of high-quality ribosome/mRNA particles assembled in RRL (rabbit reticulocyte lysate). Chimaerical mRNA-DNA molecules, consisting of the full-length mRNA ligated to a biotinylated desoxy-oligonucleotide, are immobilized on streptavidin-coated beads and incubated with RRL to form initiation complexes. After a washing step, the complexes are eluted by specific DNase I digestion of the DNA moiety of the chimaera, releasing initiation complexes in native conditions. Using this simple and robust purification setup, 80S particles properly programmed with full-length histone H4 mRNA were isolated with the expected ribosome/mRNA molar ratio of close to 1. We show that by using this novel approach purified ribosomal particles can be obtained that are suitable for biochemical and structural studies, in particular single-particle cryo-EM (cryo-electron microscopy). This purification method thus is a versatile tool for the isolation of fully functional RNA-binding proteins and macromolecular RNPs.
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