A practical approach to isolate 48S complexes: affinity purification and analyses.
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.
Locker
Nicolas
N
MRC Laboratory of Molecular Biology, Cambridge, United Kingdom.
Lukavsky
Peter J
PJ
D000818:Animals; D052157:Aptamers, Nucleotide / Q000138:chemical synthesis; D002457:Cell Extracts; D002474:Cell-Free System; D002499:Centrifugation, Density Gradient; D002846:Chromatography, Affinity / Q000379:methods*; D039642:Eukaryotic Initiation Factors / Q000302:isolation & purification* / Q000378:metabolism*; D015151:Immunoblotting; D010442:Peptide Chain Initiation, Translational; D012333:RNA, Messenger / Q000138:chemical synthesis*; D011817:Rabbits; D012156:Reticulocytes / Q000378:metabolism; D013307:Streptomycin / Q000737:chemistry
D052157:Aptamers, Nucleotide; D002457:Cell Extracts; D039642:Eukaryotic Initiation Factors; D012333:RNA, Messenger; D013307:Streptomycin