CANONICAL AND NON‐CANONICAL MECHANISM OF TRANSLATION INITIATION IN EUKARYOTES

Translation initiation consists of linked stages during which initiator tRNA, 40S and 60S ribosomal subunits are assembled by eukaryotic initiation factors (eIFs) into an 80S ribosome at the initiation codon of mRNA. These stages were reconstituted in vitro on different mRNAs from individual purified components to analyze the 5′ end-dependent mechanism of initiation and distinct non-canonical mechanisms of 5′ end-independent initiation. Initiation on most mRNAs begins with binding of eIF3 and an initiator tRNA/eIF2-GTP complex to the 40S subunit. eIF4A, 4B and 4F recruited the resulting 43S complex to the 5′-proximal region of mRNA but ribosomal scanning to the initiation codon required eIFs 1 and 1A. Displacement of factors from the resulting 48S complex and joining of a 60S subunit to form an 80S ribosome required eIF5-mediated hydrolysis of eIF2-bound GTP and eIF5B, which has an essential a ribosome-dependent GTPase activity. Analysis of internal ribosomal entry site (IRES)-mediated initiation on various viral mRNAs revealed distinct mechanisms with progressively simpler factor requirements. Each involved specific, non-canonical interactions with canonical components of the translation apparatus. Initiation on the 450nt.-long Encephalomyocarditis virus IRES involved specific binding of eIF4G/4A to the IRES followed by ATP-dependent recruitment of the 43S complex to the AUG codon, initiation on the hepatitis C virus IRES occurred by direct binding of the 43S complex to the AUG codon, and initiation on the 200nt.-long Cricket paralysis virus IRES occurred by ribosomal binding to the start site independently of factors and initiator tRNA. Supported by funds from NIH, NSF, HHMI & NATO