In Vitro Reconstitution of Eukaryotic Translation Reveals Cooperativity between Release Factors eRF1 and eRF3

SummaryEukaryotic translation termination is triggered by peptide release factors eRF1 and eRF3. Whereas eRF1 recognizes all three termination codons and induces hydrolysis of peptidyl tRNA, eRF3's function remains obscure. Here, we reconstituted all steps of eukaryotic translation in vitro using purified ribosomal subunits; initiation, elongation, and termination factors; and aminoacyl tRNAs. This allowed us to investigate termination using pretermination complexes assembled on mRNA encoding a tetrapeptide and to propose a model for translation termination that accounts for the cooperative action of eRF1 and eRF3 in ensuring fast release of nascent polypeptide. In this model, binding of eRF1, eRF3, and GTP to pretermination complexes first induces a structural rearrangement that is manifested as a 2 nucleotide forward shift of the toeprint attributed to pretermination complexes that leads to GTP hydrolysis followed by rapid hydrolysis of peptidyl tRNA. Cooperativity between eRF1 and eRF3 required the eRF3 binding C-terminal domain of eRF1

Quantitative analysis of ribosome–mRNA complexes at different translation stages

Inhibition of primer extension by ribosome–mRNA complexes (toeprinting) is a proven and powerful technique for studying mechanisms of mRNA translation. Here we have assayed an advanced toeprinting approach that employs fluorescently labeled DNA primers, followed by capillary electrophoresis utilizing standard instruments for sequencing and fragment analysis. We demonstrate that this improved technique is not merely fast and cost-effective, but also brings the primer extension inhibition method up to the next level. The electrophoretic pattern of the primer extension reaction can be characterized with a precision unattainable by the common toeprint analysis utilizing radioactive isotopes. This method allows us to detect and quantify stable ribosomal complexes at all stages of translation, including initiation, elongation and termination, generated during the complete translation process in both the in vitro reconstituted translation system and the cell lysate. We also point out the unique advantages of this new methodology, including the ability to assay sites of the ribosomal complex assembly on several mRNA species in the same reaction mixture