Eukaryotic initiation factor (eIF) 4G coordinates ribosomal binding and subsequent ribosomal scanning on the 5'NTR of an mRNA by binding to factors that are required for both steps. eIF4G is a subunit of eIF4F, which also has eIF4E (cap-binding protein) and eIF4A (helicase) subunits. This project will characterize the roles of eIF4G and associated factors in these two stages of initiation using mRNAs that depend on either the 5'-terminal cap or an internal ribosomal entry site (IRES) for ribosomal binding. Loading of the 40S ribosomal subunit onto mRNA may involve local unwinding of mRNA by bound eIF4F and interaction between eIF4G and ribosome-associated eIF3. The roles of eIF4G's interactions with RNA, eIF4A and eIF3 in ribosomal attachment and scanning will be characterized by integrating biochemical assays (such as eIF4G's influence on eIF4A's helicase activity) with reconstitution of initiation on model mRNAs. The role of Ded1p, another RNA helicase that has been implicated in scanning will also be characterized. Initiation on the encephalomyocarditis virus (EMCV) IRES requires specific binding by eIF4G/4A. The mechanisms of recognition of this IRES by eIF4G/4A, of enhancement of binding of eIF4G/4A to related IRESs by specific IRES trans-acting factors, and of promotion of ribosomal attachment by the eIF4G/4A/IRES complex will be determined. The cap-binding activity of eIF4F is down-regulated during mitosis and apoptosis when some cellular IRES-containing mRNAs remain active. The hypothesis that these IRESs resemble EMCV in interacting with eIF4G/4A to promote ribosomal binding when eIF4E is inactive will be tested. These studies will provide insights into mechanisms for selective translation of specific mRNAs.
