The integrated stress response (ISR) is crucial for cellular adaptation to various stress conditions, involving the phosphorylation of eIF2α and translational control to restore protein homeostasis. The eIF2α kinase GCN2 plays a key role in this process, being activated by amino acid depletion and other stress conditions. Two main mechanisms are proposed to trigger GCN2 activation: accumulation of uncharged tRNAs and ribosome collisions. The study investigates the role of these mechanisms in GCN2 activation under different stress conditions. Results show that while ribosome collisions are essential for GCN2 activation in response to translational elongation inhibitors, conditions that trigger deacylation of tRNAs activate GCN2 through direct association with affected tRNAs. Both mechanisms require the GCN2 regulatory domain related to histidyl tRNA synthetases. UV irradiation, which depletes amino acids and increases uncharged tRNAs, activates GCN2 without requiring ribosome collisions. The findings indicate that multiple mechanisms activate GCN2 during diverse stresses, with some involving direct engagement with uncharged tRNAs and others requiring ribosome collisions.The integrated stress response (ISR) is crucial for cellular adaptation to various stress conditions, involving the phosphorylation of eIF2α and translational control to restore protein homeostasis. The eIF2α kinase GCN2 plays a key role in this process, being activated by amino acid depletion and other stress conditions. Two main mechanisms are proposed to trigger GCN2 activation: accumulation of uncharged tRNAs and ribosome collisions. The study investigates the role of these mechanisms in GCN2 activation under different stress conditions. Results show that while ribosome collisions are essential for GCN2 activation in response to translational elongation inhibitors, conditions that trigger deacylation of tRNAs activate GCN2 through direct association with affected tRNAs. Both mechanisms require the GCN2 regulatory domain related to histidyl tRNA synthetases. UV irradiation, which depletes amino acids and increases uncharged tRNAs, activates GCN2 without requiring ribosome collisions. The findings indicate that multiple mechanisms activate GCN2 during diverse stresses, with some involving direct engagement with uncharged tRNAs and others requiring ribosome collisions.