This study dissects the roles of two isoforms of eIF4G, eIF4G1 and eIF4G2, by combining in vitro luciferase assays on 48 putative transcripts with computational analysis. Unexpectedly, most putative eIF4G1- associated transcripts maintained or increased translation following eIF4G1 knockdown, suggesting that their translation can persist through alternative initiation routes when eIF4G1 levels are reduced. Similarly, a majority of putative eIF4G2-associated transcripts retained or increased translation following eIF4G2 knockdown, while a smaller subset exhibited reduced translation consistent with stronger eIF4G2 dependence. Notably, the addition of recombinant Nat1—the murine ortholog of human eIF4G2 (also known as DAP5/p97), a protein implicated in non-canonical translation initiation—partially altered translation output in a subset of eIF4G2-associated conditions, though no statistically significant global rescue relative to eIF4G2 knockdown was observed. Comprehensive motif discovery revealed a highly conserved GC-rich 16-nucleotide sequence whose composition is consistent with RNA G-quadruplex- forming sequences. Together, these results suggest that eIF4G isoforms act as context-dependent regulators of translational plasticity, with potential implications for stem cell biology and translational dysregulation in disease.