A reconstituted cell-free assay for the evaluation of the intrinsic activity of purified human ribosomes

Abstract

We describe a cell-free translation system for evaluating the activity of ribosomes stringently purified from human cells. This system is based on in vitro reconstitution of the cellular translation machinery, in which a ribosome-free rabbit reticulocyte lysate (RRL) is reassembled with human ribosomes and in vitro-transcribed reporter mRNAs. The protocol describes the preparation of the RRL-derived fractions, purification of ribosomes devoid of detectable nonribosomal-associated factors, and assembly of the reactions to evaluate ribosomal translational efficiency and fidelity using appropriate reporter transcripts. The whole procedure can be completed in ∼2.5 d (plus 2 weeks for RRL preparation and cell expansion time). This protocol can be applied to study intrinsic functional properties (cis-acting element-mediated translation initiation or translational fidelity) of ribosome populations from different sources (including nonhuman origin). It is therefore useful for the characterization of ribosomal function in ribosomopathies and cancer, and it will be applicable in the emerging fields of ribosome diversity and specialized ribosomes.