The wheat germ protein synthesis system

Abstract

Publisher Summary This chapter describes the preparation of the cell-free extract, a typical translation assay, and procedures for identifying translational products. Cell-free extracts of wheat germ catalyze the incorporation of radioactive amino acids into protein in the presence of exogenously supplied messenger RNA (mRNA). Three types of assays are used to analyze the levels of different factors. The first type of assay includes the tobacco mosaic virus (TMV)-RNA-dependent incorporation of amino acid that serves as the primary criteria for establishing the function of a particular component in protein synthesis and the poly (U)-dependent polymerization of phenylalanine that identifies a particular component as an elongation factor. The second groups of assay include the formation of factor · Met - tRNA Met i complexes that identifies the protein components involved in the formation of ternary complexes with Guanosine-5'-triphosphate (GTP) and the formation of ribosome · Met - tRNA Met i complexes that determine the components participating in the transfer of the · Met - tRNA Met i complex to the 40 S ribosome subunit. The third type of assay measures the binding of radioactive mRNA to ribosomes and identifies the components involved in this reaction.