Differential requirements for P stalk components in activating yeast protein kinase Gcn2 by stalled ribosomes during stress

The General Amino Acid Control is a conserved response to amino acid starvation involving activation of protein kinase Gcn2, which phosphorylates eukaryotic initiation factor 2 (eIF2α) with attendant inhibition of global protein synthesis and increased translation of yeast transcriptional activator GCN4. Gcn2 can be activated by either amino acid starvation or conditions that stall elongating ribosomes without reducing aminoacylation of tRNA, but it is unclear whether distinct molecular mechanisms operate in these two circumstances. We identified three regimes that activate Gcn2 in yeast cells by starvation-independent (SI) ribosome-stalling: treatment with tigecycline, eliminating the sole gene encoding tRNA(Arg)(UCC), and depletion of translation termination factor eRF1. We further demonstrated requirements for the tRNA- and ribosome-binding domains of Gcn2, the positive effector proteins Gcn1/Gcn20, and the tethering of at least one of two distinct P1/P2 heterodimers to the uL10 subunit of the ribosomal P stalk, for detectable activation by SI-ribosome stalling. Remarkably, no tethered P1/P2 proteins were required for strong Gcn2 activation elicited by starvation for histidine or branched-chain amino acids isoleucine/valine. These results indicate that Gcn2 activation has different requirements for the P stalk depending on how ribosomes are stalled. We propose that accumulation of deacylated tRNAs in amino acid-starved cells can functionally substitute for the P stalk in binding to the histidyl-tRNA synthetase-like domain of Gcn2 for eIF2α kinase activation by ribosomes stalled with A sites devoid of the eEF1A∙GTP∙aminoacyl–tRNA ternary complex.