Activation of eIF2alpha kinase GCN2 requires GCN1-ribosome interaction and attendant modulation of A-site function by GCN1.
Evelyn Sattlegger, Alan G Hinnebusch
National Institutes of Health, NICHD, 9000 Rockville Pike, Bethesda, MD, 20892-2427, USA
Translation initiation factor 2 (eIF2) delivers tRNAiMet to the initiating ribosome. Phosphorylation of the alpha subunit of eIF2 by the protein kinase GCN2 is required to down-regulate protein synthesis in response to amino acid starvation and to specifically stimulate translation of GCN4, the transcriptional activator of amino acid biosynthetic enzymes subject to general amino acid control. GCN2 is activated by uncharged tRNAs that accumulate in starved cells, and we have proposed that GCN1 facilitates transfer of uncharged tRNA from the ribosomal decoding (A) site to GCN2 for kinase activation. Here we provide the first evidence that GCN1-ribosome interaction is essential for GCN2 activation. Putative ribosome binding domains in GCN1 reside in the elongation factor 3-like region, and in a region with a predicted amphipathic alpha helix. We found that mutations in both regions lead to decreased ribosome binding by GCN1 and impair the activation of GCN2. In addition, mutation in the alpha helix diminished sensitivity to the drug paromomycin, which reduces translation fidelity by binding to the A-site. These data support our model that GCN1 modulates A-site function to promote transfer of uncharged tRNAs from an elongating ribosome to GCN2. The fact that loss of GCN1-ribosome interaction decreases sensitivity to paromomycin suggests that yeast cells sacrifice optimal A-site function to permit codon-specific detection of uncharged tRNAs on the ribosome by GCN1.
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