Publisher Summary This chapter discusses the analysis and reconstitution of translation initiation in vitro . Translation initiation is the rate-limiting step in protein biosynthesis, and alteration of the initiation factors by covalent modification—such as phosphorylation—or by mutation can have dramatic effects on the rate of protein synthesis. The budding yeast, Saccharomyces cerevisiae ( S.cerevisiae ), provides an ideal system to investigate structure–function relationships for conserved eukaryotic translation initiation factors (eIF) by combining powerful genetic tools with biochemical analysis of cell-free extracts and purified factors. Some of the individual reactions in the initiation pathway can be assayed in yeast whole cell extracts (WCEs) or by using the relevant purified factors in model assays. The chapter describes assays using both WCEs and purified elFs. For the latter, the chapter focuses on the formation of the ternary complex (TC), the recycling of eIF2-GDP to eIF2-GTP by eIF2, and stimulation of GTP hydrolysis in the TC by eIF5 The chapter describes the use of a single extract to assay the overall rate of protein synthesis with a luciferase reporter mRNA and the ability of the endogenous eIFs to deliver Met- Met i and mRNA to the 40S ribosome.
