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Translation termination is an important step in eukaryotic protein synthesis and is mainly mediated by the release factors eRF1 and eRF3, potentially involving the ATPase ABCE1. So far, the specific role of ABCE1 in translation termination is still not fully understood. This study aimed to establish a minimal eukaryotic translation termination system and to investigate the functional relation among eRF1, eRF3, and ABCE1 during translation termination.

In this study, a pre-termination complex was assembled using Saccharomyces cerevisiae 80S ribosomes, leaderless mRNA, and BODIPY-labelled initiator Met-tRNA^fMet. The factors eRF1, eRF3, and ABCE1 were then expressed and purified from Escherichia coli. Stopped-flow fluorescence assays and phosphate release assays were performed to evaluate the dynamic response of the translation termination system and the GTPase activity of eRF3. The Pi release assay showed that eRF1 significantly enhanced the GTP hydrolysis activity of eRF3 by approximately seven times. Based on this, ABCE1 further increased the hydrolysis rate by nearly 50%.

The kinetic assays showed that the established leaderless mRNA system could successfully form a functional pre-termination complex and respond to release factors. In the stopped-flow assay, after the addition of eRF1 and eRF3, the fluorescence change rate increased from 0.003 s⁻¹ to 0.006 s⁻¹. This suggests that the system has the potential to detect peptide release. 

In conclusion, this study successfully established a minimal in vitro system for studying the kinetics of eukaryotic translation termination and provided initial evidence for the potential role of ABCE1 in regulating eRF3 activity. However, due to the background instability still present in the pre-termination complex, further optimisation of the system is required to improve system stability and detection accuracy.