The tRNA-binding moiety in GCN2 contains a dimerization domain that interacts with the kinase domain and is required for tRNA binding and kinase activation.
GCN2 stimulates translation of GCN4 mRNA in amino acid-starved cells by phosphorylating translation initiation factor 2. GCN2 is activated by binding of uncharged tRNA to a domain related to histidyl-tRNA synthetase (HisRS). The HisRS-like region contains two dimerization domains (HisRS-N and HisRS-C) required for GCN2 function in vivo but dispensable for dimerization by full-length GCN2. Residues corresponding to amino acids at the dimer interface of Escherichia coli HisRS were required for dimerization of recombinant HisRS-N and for tRNA binding by full-length GCN2, suggesting that HisRS-N dimerization promotes tRNA binding and kinase activation. HisRS-N also interacted with the protein kinase (PK) domain, and a deletion impairing this interaction destroyed GCN2 function without reducing tRNA binding; thus, HisRS-N-PK interaction appears to stimulate PK function. The C-terminal domain of GCN2 (C-term) interacted with the PK domain in a manner disrupted by an activating PK mutation (E803V). These results suggest that the C-term is an autoinhibitory domain, counteracted by tRNA binding. We conclude that multiple domain interactions, positive and negative, mediate the activation of GCN2 by uncharged tRNA.
Laboratory of Eukaryotic Gene Regulation, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892, USA.
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Qiu
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Dong
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Hu
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Francklyn
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Hinnebusch
D000494:Allosteric Regulation; D001665:Binding Sites; D004252:DNA Mutational Analysis; D019281:Dimerization; D004789:Enzyme Activation; D006643:Histidine-tRNA Ligase / Q000235:genetics; D008957:Models, Genetic; D011494:Protein Kinases / Q000235:genetics / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D012343:RNA, Transfer / Q000378:metabolism*
D012343:RNA, Transfer; D011494:Protein Kinases; D006643:Histidine-tRNA Ligase