5 Mechanism and Regulation of Initiator Methionyl-tRNA Binding to Ribosomes

In its simplest terms, the process of translation initiation in eukaryotic organisms consists of the binding of methionyl-initiator tRNA (Met-tRNA i Met ) and mRNA to the 40S ribosomal subunit, pairing of the anticodon of Met-tRNA i Met with the AUG start codon in mRNA, and joining of the 60S ribosomal subunit to form an 80S initiation complex. Each of these steps is stimulated by soluble protein factors known as eukaryotic initiation factors (eIFs). Reconstitution of this process in vitro using purified ribosomes and eIFs indicated that binding of Met-tRNA i Met to the 40S subunit is a prerequisite for mRNA binding (Benne and Hershey 1978; Trachsel and Staehelin 1979). The Met-tRNA i Met is transferred to the 40S subunit by a ternary complex consisting of Met-tRNA i Met , the heterotrimeric initiation factor 2 (eIF2), and GTP, and this reaction is stimulated by eIF3, eIF1A, and possibly eIF5B. The resulting 43S preinitiation complex binds mRNA, forming the 48S complex, in a reaction promoted by the mRNA-associated factors (eIF4E, eIF4G, eIF4A, eIF4B, and poly[A]-binding protein) and the eIF3 residing in the 43S complex. The preinitiation complex scans the mRNA, and pairing between the anticodon of Met-tRNA i Met and the AUG start codon triggers hydrolysis of GTP by eIF2, dependent of the GTPase activating protein (GAP) eIF5. After release of eIF2–GDP and eIF3, the 60S subunit joins the assembly in a reaction stimulated by eIF5B and involves the hydrolysis of a second molecule of GTP (see Chapters 2 and 9). The eIF2–GDP is inactive for binding Met-tRNA i Met and must be converted to...