The integrated stress response (ISR) is a cellular mechanism that helps cells adapt to various stress conditions by activating eIF2α kinase, which leads to the phosphorylation of eIF2α and the subsequent regulation of protein synthesis. GCN2 is a key eIF2α kinase that is activated by diverse stress conditions, including amino acid deprivation, UV irradiation, and ribosome collisions. Two mechanisms have been proposed for GCN2 activation: one involving the accumulation of uncharged tRNAs and another involving ribosome collisions. This study investigated these mechanisms in detail and found that while ribosome collisions are essential for GCN2 activation in response to translational elongation inhibitors, conditions that trigger deacylation of tRNAs activate GCN2 through its direct association with affected tRNAs. Both mechanisms require the GCN2 regulatory domain related to histidyl tRNA synthetases. GCN2 activation by UV irradiation features lowered amino acids and increased uncharged tRNAs, and UV-induced ribosome collisions are suggested to be dispensable. The study also showed that GCN2 activation by accumulating uncharged tRNAs occurs through direct binding with the GCN2 HARS-related domain, and that ribosome collisions are not required. These findings suggest that there are multiple mechanisms that activate GCN2 during diverse stresses, some of which involve direct engagement with uncharged tRNAs, while others require ribosome collisions. The study also demonstrated that specific tRNA depletion can trigger ribosome collisions and GCN2 activation, highlighting the complexity of the mechanisms involved in GCN2 activation.The integrated stress response (ISR) is a cellular mechanism that helps cells adapt to various stress conditions by activating eIF2α kinase, which leads to the phosphorylation of eIF2α and the subsequent regulation of protein synthesis. GCN2 is a key eIF2α kinase that is activated by diverse stress conditions, including amino acid deprivation, UV irradiation, and ribosome collisions. Two mechanisms have been proposed for GCN2 activation: one involving the accumulation of uncharged tRNAs and another involving ribosome collisions. This study investigated these mechanisms in detail and found that while ribosome collisions are essential for GCN2 activation in response to translational elongation inhibitors, conditions that trigger deacylation of tRNAs activate GCN2 through its direct association with affected tRNAs. Both mechanisms require the GCN2 regulatory domain related to histidyl tRNA synthetases. GCN2 activation by UV irradiation features lowered amino acids and increased uncharged tRNAs, and UV-induced ribosome collisions are suggested to be dispensable. The study also showed that GCN2 activation by accumulating uncharged tRNAs occurs through direct binding with the GCN2 HARS-related domain, and that ribosome collisions are not required. These findings suggest that there are multiple mechanisms that activate GCN2 during diverse stresses, some of which involve direct engagement with uncharged tRNAs, while others require ribosome collisions. The study also demonstrated that specific tRNA depletion can trigger ribosome collisions and GCN2 activation, highlighting the complexity of the mechanisms involved in GCN2 activation.