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Selective control of metabolism by the GCN2 eIF2 kinase pathway in Saccharomyces cerevisiae

ABSTRACT: When eukaryotic cells are deprived of amino acids, uncharged tRNAs accumulate and activate the conserved GCN2 protein kinase. We examine how yeast growth and tRNA charging or aminoacylation is affected during amino acid depletion in the presence and absence of GCN2. tRNA charging is measured using a microarray technique which allows for simultaneous measurement of all cytosolic tRNAs. A fully prototrophic and its isogenic GCN2 deletion strain were used.

ORGANISM(S): Saccharomyces cerevisiae

PROVIDER: GSE22452 | GEO | 2010/08/01

SECONDARY ACCESSION(S): PRJNA128539

REPOSITORIES: GEO

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Selective control of metabolism by the GCN2 eIF2 kinase pathway in Saccharomyces cerevisiae

Project description:When eukaryotic cells are deprived of amino acids, uncharged tRNAs accumulate and activate the conserved GCN2 protein kinase. We examine how yeast growth and tRNA charging or aminoacylation is affected during amino acid depletion in the presence and absence of GCN2. tRNA charging is measured using a microarray technique which allows for simultaneous measurement of all cytosolic tRNAs. A fully prototrophic and its isogenic GCN2 deletion strain were used. We measured relative tRNA charging levels in yeast strains with an intact and deleted GCN2.

2010-08-01 | E-GEOD-22452 | biostudies-arrayexpress

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Project description:GCN2 is a stress response kinase that phosphorylates the translation initiation factor eIF2to inhibit general protein synthesis when activated by uncharged tRNA and stalled ribosomes. The presence of a HisRS-like domain in GCN2, normally associated with the ability to bind and aminoacylate tRNAs, led to the hypothesis that eIF2 kinase activity is regulated by the direct binding of this domain to uncharged tRNA. Here we solved the structure of the HisRS-like domain in the context of full-length GCN2 by cryoEM. Structure and function analysis shows the HisRS-like domain of GCN2 has lost tRNA charging, ATP binding, and histidine binding activity but retains the ability to bind tRNA. Hydrogen deuterium exchange mass spectrometry (HX-MS), site-directed mutagenesis and computational docking experiments support a tRNA binding model that overlaps with but is partially shifted from that employed by bona fide HisRS enzymes. These results demonstrate that the HisRS-like domain of GCN2 is a pseudoenzyme and advance our understanding of GCN2 regulation and function.

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Translation in amino acid-poor environments is limited by tRNAGln charging

Project description:An inadequate supply of amino acids leads to accumulation of uncharged tRNAs, which can bind and activate GCN2 kinase to reduce translation. Here, we show that glutamine-specific tRNAs selectively become uncharged when extracellular amino acid availability is compromised. In contrast, all other tRNAs retain charging of their cognate amino acids in a manner that is dependent upon intact lysosomal function. In addition to GCN2 activation and reduced total translation, the reduced charging of tRNAGln in amino acid-deprived cells also leads to specific depletion of proteins containing polyglutamine tracts including core binding factor α1, mediator subunit 12, transcriptional coactivator CBP and TATA-box binding protein. Treating amino acid-deprived cells with exogenous glutamine or glutaminase inhibitors restores tRNAGln charging and the levels of polyglutamine-containing proteins. Together, these results demonstrate that the activation of GCN2 and the translation of polyglutamine-encoding transcripts serve as the key sensors of glutamine availability in mammalian cells.

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Project description:Summary Aminoacyl-tRNA synthetases (aaRSs) serve a dual role in charging tRNAs. Their enzymatic activities both provide protein synthesis flux and reduce uncharged tRNA levels. Although uncharged tRNAs can negatively impact bacterial growth, substantial concentrations of tRNAs remain deacylated even under nutrient-rich conditions. Here we show that tRNA charging in Bacillus subtilis is not maximized due to optimization of aaRS production during rapid growth, which prioritizes demands in protein synthesis over charging levels. The presence of uncharged tRNAs is alleviated by precisely tuned translation kinetics and the stringent response, both insensitive to aaRS overproduction but sharply responsive to underproduction, allowing for just-enough aaRS production atop a “fitness cliff”. Notably, we find that the stringent response mitigates fitness defects at all aaRS underproduction levels even without external starvation. Thus, adherence to minimal, flux-satisfying protein production drives limited tRNA charging and provides a basis for the sensitivity and setpoints of an integrated growth-control network.

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Project description:Here we develop a high throughput sequencing method that enables accurate determination of charged tRNA fractions at single base resolution (Charged tRNA-seq). Our method takes advantage of the recently developed DM-tRNA-seq method, but includes additional chemical steps that specifically remove the 3'A residue in the uncharged tRNA. Charging fraction is obtained by counting the fraction of A-ending reads versus A+C-ending reads for each tRNA species. In HEK293T cells, most cytosolic tRNAs are charged at >90% levels, whereas tRNASer and tRNAThr are generally charged at lower levels. These low charging levels were validated using acid denaturing gels. Our method should be widely applicable for investigations of tRNA charging as a parameter for biological regulation.

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McFarland2020 - Synthetase sequestration Model:a combined translation and tRNA charging cycle

Project description:The Synthetase Sequestration Model (SSM) is a simplified translation model that considers explicitly two main steps in the process of tRNA aminoacylation: first, the tRNA is bound by the aminoacyl tRNA synthetase, and in a second step, the amino acid is attached to the tRNA. The tRNA then participates in the translation reaction, becoming deacylated as a result. The tRNA exists in states bound, charged and uncharged. In the bound state, the tRNA is bound to the synthetase but uncharged, i.e., the tRNA is sequestered by the synthetase. The model predicts how the balance between the three different tRNA states (empty, bound and charged) changes depending on aminoacyl tRNA synthetase availability.

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Yeast glutamine tRNA synthetase doxycycline shut-off

Project description:During protein synthesis, charged tRNAs deliver amino acids to translating ribosomes, and are then re-charged by tRNA synthetases (aaRS). In humans, mutant aaRS cause a diversity of neurological disorders, but their molecular aetiologies are incompletely characterised. To understand system responses to aaRS depletion, the yeast glutamine aaRS gene (GLN4) was transcriptionally regulated using doxycycline by tet-off control. Depletion of Gln4p inhibited growth, and induced a transcriptional GCN4 amino acid starvation response, indicative of uncharged tRNA accumulation and Gcn2 kinase activation. The GCN4 response was confirmed using SILAC proteomics, using heavy isotope labelling to identify changes in protein expression during glutamine tRNA synthetase depletion.

2020-02-12 | PXD017140 | Pride

Tarsl2 is evolved not for tRNA charging in vivo

Project description:As a newly evolved duplicated gene of TARS1 (encoding cytoplasmic threonyl-tRNA synthetase), TARSL2 (TARS3) encodes a protein highly similar with TARS1 with only obvious difference in N-terminus. Albeit with tRNA charging activity in vitro and incorporated into the multiple tRNA synthetase complex (MSC), whether TARSL2 is able to function as a tRNA synthetase to substitute TARS1 in vivo is unclear. In this work, we found that Tars1 was essential for embryonic development. In contrast, knockout of Tarsl2 was achievable. Loss of Tarsl2 had no effect on both abundance and charging levels of all tRNAThr isoacceptors, indicating Tarsl2 is not for tRNAThr metabolism. Furthermore, Tarsl2 deletion did not influence protein levels of other MSC components, MSC integrity and aminoacylation levels of non-cognate tRNAs by MSC components. However, a severe retardation of mice development was observed after 3 weeks accompanied with an elevated metabolism capacity and muscle development abnormity. We further constructed a Tarsl2-deleted zebrafish, which exhibited no change in the level and charging of tRNAThrs. Collectively, these data suggest that Tarsl2 is evolved not for canonical tRNAThr aminoacylation and cannot substitute Tars1 in vivo.

2023-05-31 | GSE218318 | GEO

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