Document Detail


Rapid purification of ribosomal particles assembled on histone H4 mRNA: a new method based on mRNA-DNA chimeras.
MedLine Citation:
PMID:  23140180     Owner:  NLM     Status:  Publisher    
Abstract/OtherAbstract:
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. Here we present a novel method designed for the purification of high-quality ribosome/mRNA particles assembled in rabbit reticulocyte lysate (RRL). Chimerical 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 chimera, 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 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 electron microscopy (cryo-EM). This purification method thus is a versatile tool for the isolation of full, functional RNA-binding proteins and macromolecular RNPs.
Authors:
Lydia Prongidi-Fix; Laure Schaeffer; Angelita Simonetti; Sharief Barends; Jean-François Ménétret; Bruno Klaholz; Gilbert Eriani; Franck Martin
Publication Detail:
Type:  JOURNAL ARTICLE     Date:  2012-11-12
Journal Detail:
Title:  The Biochemical journal     Volume:  -     ISSN:  1470-8728     ISO Abbreviation:  Biochem. J.     Publication Date:  2012 Nov 
Date Detail:
Created Date:  2012-11-12     Completed Date:  -     Revised Date:  -    
Medline Journal Info:
Nlm Unique ID:  2984726R     Medline TA:  Biochem J     Country:  -    
Other Details:
Languages:  ENG     Pagination:  -     Citation Subset:  -    
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