Genome-wide analysis of tRNA charging and activation of the eIF2 kinase Gcn2p.
When cells are subjected to nutritional stress, uncharged tRNAs accumulate and activate Gcn2p phosphorylation of eukaryotic initiation factor-2 (eIF2) and the general amino acid control pathway. The Gcn2p regulatory domain homologous to histidyl-tRNA synthetases is proposed to bind to uncharged tRNA, directly contributing to activation of Gcn2p. Here we apply a microarray technology to analyze genome-wide changes in tRNA charging in yeast upon activation of Gcn2p in response to amino acid starvation and high salinity, a stress not directly linked to nutritional deficiency. This microarray technology is applicable for all eukaryotic cells. Strains were starved for histidine, leucine, or tryptophan and shown to rapidly induce Gcn2p phosphorylation of eIF2. The relative charging level of all tRNAs was measured before and after starvation, and Gcn2p activation and the intracellular levels of the starved amino acid correlate with the observed decrease in tRNA charging. Interestingly, in some cases, tRNAs not charged with the starved amino acid became deacylated more rapidly than tRNAs charged with the starved amino acid. This increase in uncharged tRNA levels occurred although the intracellular levels for these non-starved amino acids remained unchanged. Additionally, treatment of a wild-type strain with high salinity stress showed transient changes in the charging of several different tRNAs. These results suggest that Gcn2p can be activated by many different tRNA species in the cell. These results also depict a complex cellular relationship between tRNA charging, amino acid availability, and non-nutrient stress. These relationships are best revealed by simultaneous monitoring of the charging level of all tRNAs.
Zaborske
John M
JM
Department of Biochemistry and Molecular Biology, University of Chicago, Chicago, Illinois 60637, USA.
Narasimhan
Jana
J
Jiang
Li
L
Wek
Sheree A
SA
Dittmar
Kimberly A
KA
Freimoser
Florien
F
Pan
Tao
T
Wek
Ronald C
RC
D000596:Amino Acids / Q000737:chemistry; D015152:Blotting, Northern; D015852:Eukaryotic Initiation Factor-2 / Q000378:metabolism; D016681:Genome, Fungal; D015151:Immunoblotting; D007930:Leucine / Q000737:chemistry; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000502:physiology*; D012343:RNA, Transfer / Q000737:chemistry; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000502:physiology*; D012965:Sodium Chloride / Q000494:pharmacology; D013997:Time Factors; D014364:Tryptophan / Q000737:chemistry; D019892:eIF-2 Kinase / Q000378:metabolism*
D000596:Amino Acids; D015852:Eukaryotic Initiation Factor-2; D029701:Saccharomyces cerevisiae Proteins; D012965:Sodium Chloride; D014364:Tryptophan; D012343:RNA, Transfer; C512912:GCN2 protein, S cerevisiae; D017346:Protein Serine-Threonine Kinases; D019892:eIF-2 Kinase; D007930:Leucine