Yeast reconstituted translation assays for analysis of eIF5A function

Polyamines are critically important for protein synthesis. Through their positive ionic charge, polyamines readily bind to ribosomes, as well as to mRNAs and tRNAs. Moreover, the polyamine spermidine serves as a substrate for the synthesis of hypusine, an essential post-translational modification on the translation factor eIF5A. Though originally thought to function in translation initiation, eIF5A is now known to generally promote translation elongation and termination. Moreover, translation of certain motifs like polyproline show a greater dependency on eIF5A. In this chapter, we describe the biochemical assays we use to study eIF5A and its regulation. Owing to the complex nature of protein synthesis, these assays require the purification of over 10 translation factors plus ribosomes, tRNAs, and aminoacyl-tRNA synthetases. We describe the methods used to purify these components, to synthesize the mRNA templates for translation, and to resolve the translation products by electrophoretic thin-layer chromatography. With the recent identification of eIF5A as a key target for regulating the synthesis of polyamine synthesis and transport, and the recent identification of mutations in eIF5A causing a neurodevelopmental disorder, the assays described in this chapter will be useful in further elucidating the function and regulation of this enigmatic protein.