Running title: eIF4GI or DAP5 drives cap-independent translation initiationKeywords: 5' CITE | IRES | eIF4GI | DAP5 | fluorescence anisotropy AbstractDuring unfavorable human cellular conditions (e.g., tumor hypoxia, viral infection, etc.), canonical, cap-dependent mRNA translation is suppressed. Nonetheless, a subset of physiologically important mRNAs (e.g., HIF-1α, FGF-9, and p53) is still translated by an unknown, capindependent mechanism. Additionally, expression levels of eIF4G and its homolog, death associated protein 5 (DAP5), are elevated. Using fluorescence anisotropy binding studies, luciferase reporter-based in vitro translation assays, and mutational analyses, here we demonstrate that eIF4GI and DAP5 specifically bind to the 5' UTRs of these cap-independently translated mRNAs. Surprisingly, we find that the eIF4E binding domain of eIF4GI increases not only the binding affinity, but also the selectivity among these mRNAs. We further demonstrate that the affinities of eIF4GI and DAP5 binding to these 5' UTRs correlate with the efficiency with which these factors drive cap-independent translation of these mRNAs. Integrating the results of our binding and translation assays, we show that eIF4GI and/or DAP5 are critical for recruitment of a specific subset of mRNAs to the ribosome and provide mechanistic insight into their capindependent translation. Running title: eIF4GI or DAP5 drives cap-independent translation initiationKeywords: 5' CITE | IRES | eIF4GI | DAP5 | fluorescence anisotropy