Uncharged tRNA activates GCN2 by displacing the protein kinase moiety froma bipartite tRNA-Binding domain

Protein kinase GCN2 regulates translation in amino acid-starved cells by ph osphorylating eIF2. GCN2 contains a regulatory domain related to histidyl-t RNA synthetase (HisRS) postulated to bind multiple deacylated tRNAs as a ge neral sensor of starvation. In accordance with this model, GCN2 bound sever al deacylated tRNAs with similar affinities, and aminoacylation of tRNA(Phe ) weakened its interaction with GCN2. Unexpectedly, the C-terminal ribosome binding segment of GCN2 (C-term) was required in addition to the HisRS dom ain for strong tRNA binding. A combined HisRS+ C-term segment bound to the isolated protein kinase (PK) domain in vitro, and tRNA impeded this interac tion. An activating mutation (GCN2(C)-E803V) that weakens PK-C-term associa tion greatly enhanced tRNA binding by GCN2. These results provide strong ev idence that tRNA stimulates the GCN2 kinase moiety by preventing an inhibit ory interaction with the bipartite tRNA binding domain.