Uncharged tRNA activates GCN2 by displacing the protein kinase moiety from a bipartite tRNA-binding domain.
Protein kinase GCN2 regulates translation in amino acid-starved cells by phosphorylating elF2. GCN2 contains a regulatory domain related to histidyl-tRNA synthetase (HisRS) postulated to bind multiple deacylated tRNAs as a general sensor of starvation. In accordance with this model, GCN2 bound several deacylated tRNAs with similar affinities, and aminoacylation of tRNAphe weakened its interaction with GCN2. Unexpectedly, the C-terminal ribosome binding segment of GCN2 (C-term) was required in addition to the HisRS domain for strong tRNA binding. A combined HisRS+ C-term segment bound to the isolated protein kinase (PK) domain in vitro, and tRNA impeded this interaction. An activating mutation (GCN2c-E803V) that weakens PK-C-term association greatly enhanced tRNA binding by GCN2. These results provide strong evidence that tRNA stimulates the GCN2 kinase moiety by preventing an inhibitory interaction with the bipartite tRNA binding domain.
Dong
J
J
Laboratory of Eukaryotic Gene Regulation, National Institute of Child Health and Human Development, Bethesda, Maryland 20892, USA.
Qiu
H
H
Garcia-Barrio
M
M
Anderson
J
J
Hinnebusch
A G
AG
D001665:Binding Sites; D004268:DNA-Binding Proteins / Q000378:metabolism; D015852:Eukaryotic Initiation Factor-2 / Q000378:metabolism; D005786:Gene Expression Regulation; D006643:Histidine-tRNA Ligase / Q000737:chemistry; D008239:Lysine; D014176:Protein Biosynthesis; D011494:Protein Kinases / Q000737:chemistry* / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases; D012345:RNA, Transfer, Amino Acid-Specific / Q000378:metabolism*; D012360:RNA, Transfer, Phe / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012270:Ribosomes / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D029701:Saccharomyces cerevisiae Proteins
D004268:DNA-Binding Proteins; D015852:Eukaryotic Initiation Factor-2; D012345:RNA, Transfer, Amino Acid-Specific; D012360:RNA, Transfer, Phe; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; D011494:Protein Kinases; C512912:GCN2 protein, S cerevisiae; D017346:Protein Serine-Threonine Kinases; D006643:Histidine-tRNA Ligase; D008239:Lysine