In vitro reconstitution of translational arrest pathways.

Protein translation is tightly regulated to ensure high-fidelity expression of genetic information. Various conditions cause ribosomes to stall while synthesizing new proteins. Different types of translational arrest initiate specific mRNA surveillance, protein quality control, and stress response pathways that directly impact gene expression and protein homeostasis. Our understanding of these pathways is greatly enhanced by reconstituting these processes in cell-free systems. The high degree of biochemical manipulability of in vitro systems facilitates the identification of key machineries, mechanistic dissection of their functional roles, and structural analysis of intermediate complexes. Here, we describe principles and methods for reconstituting and analyzing translational arrest pathways in cell-free translation systems using rabbit reticulocyte lysate as an example. These approaches can be exploited to dissect various fundamental, regulatory, and quality control mechanisms of eukaryotic protein translation.