Reconstitution of Mammalian 48S Ribosomal Translation Initiation Complex

Abstract Initiation of translation is defined as the process by which a 40S ribosomal subunit, containing bound initiator methionyl-tRNA (Met-tRNA i ), is positioned at the initiation AUG codon of an mRNA to form the 48S initiation complex. Subsequently, a 60S ribosomal subunit joins the 48S initiation complex to form an elongation-competent 80S initiation complex. By use of highly purified eukaryotic translation initiation factors (eIFs), ribosomes, Met-tRNA i , mRNA, GTP as an effector molecule, and ATP as a source of energy, the initiation step of translation can be efficiently reconstituted. In this chapter, we describe the detailed procedure for efficient binding of Met-tRNA i to the 40S ribosomal subunit, the subsequent binding of the resulting 43S preinitiation complex to an mRNA, and scanning and positioning of the 43S complex at the AUG start codon of the mRNA to form the 48S initiation complex.