Abstract:
:In vitro assembly of eukaryotic translation initiation complexes requires purification of ribosomal subunits, eukaryotic initiation factors, and initiator tRNA from natural sources and therefore yields only limited material for functional and structural studies. In this chapter, we describe a robust, affinity chromatography-based method for the isolation of eukaryotic 48S initiation complexes from rabbit reticulocyte lysate (RRL). Both canonical and internal ribosome entry site (IRES)-containing mRNAs labeled with a streptomycin aptamer sequence at the 3' end can be used to purify milligram quantities of 48S particles in a simple, two-step procedure. The 48S complexes purified with this method are properly assembled at the initiation codon, contain the expected RNA and protein components in a 1:1 stoichiometry, and are functional intermediates along the initiation pathway.
journal_name
Methods Enzymoljournal_title
Methods in enzymologyauthors
Locker N,Lukavsky PJdoi
10.1016/S0076-6879(07)29005-6subject
Has Abstractpub_date
2007-01-01 00:00:00pages
83-104eissn
0076-6879issn
1557-7988pii
S0076-6879(07)29005-6journal_volume
429pub_type
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