Expression of amino acid biosynthetic genes in yeast is stimulated in response to amino acid starvation by induction of the transcriptional activator GCN4. Translation of GCN4 mRNA is induced in starved cells by phosphorylation of translation initiation factor 2 (eIF2) by protein kinase GCN2. GCN2 is activated by binding of uncharged tRNA to a histidyl- tRNA synthetase (HisRS)-related region located adjacent to the kinase domain. GCN2 regulation involves dimerization by the kinase domain, HisRS region and a C-terminal ribosome-binding segment. The latter two interactions, at least, appear to be critical for GCN2 function in vivo, although only the C-term is required for dimerization per se by GCN2. These GCN2 domains also interact with one another, and discrete segments mediating these interactions have been identified. GCN2 appears to dimerize constitutively, and we propose that tRNA binding elicits a conformational rearrangement that stimulates kinase activity of the GCN2 dimer. The positive effectors of GCN2, known as GCN1 and GCN20, are related to translation elongation factor EF3 and also have ribosome-binding activities. GCN1/GCN20 binds directly to GCN2 and discrete segments of GCN2 and GCN1 required for this interaction have been identified. We propose that GCN2 interacts with the GCN1/GCN20 complex to facilitate binding of uncharged tRNA present on the ribosome to the HisRS domain in GCN2. Phosphorylation of eIF2 by GCN2 inhibits the 5-subunit guanine nucleotide exchange factor for eIF2, known as eIF2B, preventing formation of the ternary complex with initiator tRNA (eIF2/GTP/tRNAiMet) that transfers tRNAiMet to the 40S ribosome. Three eIF2B subunits (GCN3, GCD7, and GCD2) comprise a regulatory subcomplex that binds phosphorylated eIF2 and mediates inhibition of the GCD1/GCD6 catalytic subcomplex of eIF2B. There appear to be separate binding sites in the beta subunit of eIF2 for GCD2 and GCD6, important presumably for eIF2B regulation and nucleotide exchange, respectively. Initiation factor 3 stimulates binding of the ternary complex and mRNA to the 40S ribosome. Yeast eIF3 consists of 5 conserved subunits which can form several stable subcomplexes in vivo, one of which interacts with initiation factors eIF5 and eIF1 via the NIP1 subunit of eIF3. eIF5, a GTPase activating factor for eIF2, and eIF1 are both implicated in stringent selection of AUG as start codon, and this function may depend on their mutual interaction with eIF3-NIP1. Both eIF5 and the catalytic subunit of eIF2B (GCD6) utilize conserved binding domains to interact with the N-terminal half of eIF2, their common substrate. GCD10 and GCD14 are components of a nuclear complex that is required for methylation of a specific residue in multiple tRNAs. This modification is essential for processing and accumulation of tRNAiMet, and thus ternary complex formation and translation initiation. The transcriptional activation domain in GCN4 contains hydrophobic clusters that mediate independent physical interactions with subunits of the SRB/mediator of RNA Polymerase II holoenzyme, the SPT/ADA/GCN5 (SAGA) complex, and the SWI/SNF complex. Transposon insertion mutagenesis of the yeast genome reveals that all three coactivator complexes are required for transcriptional activation by GCN4 in vivo. - YEAST/AMINO ACIDS/TRANSLATION/EIF2/PHOSPHORYLATION/KINASE/ TRANSCRIPTION/SAGA/SWI/MEDIATOR
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