The Roles of eIF4G and Associated Factors in Ribosomal Attachment and Scanning During Translation Initiation

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Eukaryotic initiation factor (eIF) 4G coordinates ribosomal binding and subsequent ribosomalscanning on the 5'NTR of an mRNA by binding to factors that are required for both steps. eIF4G is asubunit of eIF4F, which also has eIF4E (cap-binding protein) and eIF4A (helicase) subunits. This projectwill characterize the roles of eIF4G and associated factors in these two stages of initiation using mRNAsthat depend on either the 5'-terminal cap or an internal ribosomal entry site (IRES) for ribosomalbinding. Loading of the 40S ribosomal subunit onto mRNA may involve local unwinding of mRNA bybound eIF4F and interaction between eIF4G and ribosome-associated eIF3. The roles of eIF4G'sinteractions with RNA, eIF4A and eIF3 in ribosomal attachment and scanning will be characterized byintegrating biochemical assays (such as eIF4G's influence on eIF4A's helicase activity) withreconstitution of initiation on model mRNAs. The role of Ded1p, another RNA helicase that has beenimplicated 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, ofenhancement of binding of eIF4G/4A to related IRESs by specific IRES trans-acting factors, and ofpromotion of ribosomal attachment by the eIF4G/4A/IRES complex will be determined. The cap-bindingactivity of eIF4F is down-regulated during mitosis and apoptosis when some cellular IRES-containingmRNAs remain active. The hypothesis that these IRESs resemble EMCV in interacting with eIF4G/4A topromote ribosomal binding when eIF4E is inactive will be tested. These studies will provide insightsinto mechanisms for selective translation of specific mRNAs.