The molecular aetiology of tRNA synthetase depletion: induction of a GCN4 amino acid starvation response despite homeostatic maintenance of charged tRNA levels

Abstract 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 GCN4 amino acid starvation response, indicative of uncharged tRNA accumulation and Gcn2 kinase activation. Using a global model of translation that included aaRS recharging, Gln4p depletion was simulated, confirming slowed translation. Modelling also revealed that Gln4p depletion causes negative feedback that matches translational demand for Gln-tRNAGln to aaRS recharging capacity. This maintains normal charged tRNAGln levels despite Gln4p depletion, confirmed experimentally using tRNA Northern blotting. Model analysis resolves the paradox that Gln4p depletion triggers a GCN4 response, despite maintenance of tRNAGln charging levels, revealing that normally, the aaRS population can sequester free, uncharged tRNAs during aminoacylation. Gln4p depletion reduces this sequestration capacity, allowing uncharged tRNAGln to interact with Gcn2 kinase. The study sheds new light on mutant aaRS disease aetiologies, and explains how aaRS sequestration of uncharged tRNAs can prevent GCN4 activation under non-starvation conditions.

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The histidyl-tRNA synthetase-related sequence in the eIF-2 alpha protein kinase GCN2 interacts with tRNA and is required for activation in response to starvation for different amino acids.

Molecular and Cellular Biology ◽

10.1128/mcb.15.8.4497 ◽

1995 ◽

Vol 15 (8) ◽

pp. 4497-4506 ◽

Cited By ~ 274

Author(s):

S A Wek ◽

S Zhu ◽

R C Wek

Keyword(s):

Amino Acids ◽

Amino Acid ◽

Protein Kinase ◽

Trna Synthetase ◽

Amino Acid Starvation ◽

Related Sequence ◽

General Amino Acid Control ◽

Trna Synthetases ◽

Acid Control ◽

Direct Regulator

Protein kinase GCN2 is a multidomain protein that contains a region homologous to histidyl-tRNA synthetases juxtaposed to the kinase catalytic moiety. Previous studies have shown that in response to histidine starvation, GCN2 phosphorylates eukaryotic initiation factor 2 (eIF-2), to induce the translational expression of GCN4, a transcriptional activator of genes subject to the general amino acid control. It was proposed that the synthetase-related sequences of GCN2 stimulate the activity of the kinase by interacting directly with uncharged tRNA that accumulates during amino acid limitation. In addition to histidine starvation, expression of GCN4 is also regulated by a number of other amino acid limitations. Questions that we posed in this report are whether uncharged tRNA is the most direct regulator of GCN2 and whether the function of this kinase is required to recognize each of the different amino acid starvation signals. We show that GCN2 phosphorylation of eIF-2, and the resulting general amino acid control pathway, is stimulated in response to starvation for each of several different amino acids, in addition to histidine limitation. Cells containing a defective aminoacyl-tRNA synthetase also stimulated GCN2 phosphorylation of eIF-2 in the absence of amino acid starvation, indicating that uncharged tRNA levels are the most direct regulator of GCN2 kinase. Using a Northwestern blot (RNA binding) assay, we show that uncharged tRNA can bind to the synthetase-related domain of GCN2. Mutations in the motif 2 sequence conserved among class II synthetases, including histidyl-tRNA synthetases, impair the ability of this synthetase-related domain to bind tRNA and abolish GCN2 phosphorylation of eIF-2 required to stimulate the general amino acid control response. These in vivo and in vitro experiments indicate that synthetase-related sequences regulate GCN2 kinase function by monitoring the levels of multiple uncharged tRNAs that accumulate during amino acid limitations.

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Did Amino Acid Side Chain Reactivity Dictate the Composition and Timing of Aminoacyl-tRNA Synthetase Evolution?

Genes ◽

10.3390/genes12030409 ◽

2021 ◽

Vol 12 (3) ◽

pp. 409

Author(s):

Tamara L. Hendrickson ◽

Whitney N. Wood ◽

Udumbara M. Rathnayake

Keyword(s):

Amino Acids ◽

Amino Acid ◽

Genetic Code ◽

Chemical Reactivity ◽

Trna Synthetase ◽

Amino Acid Side Chain ◽

Standard Genetic Code ◽

Last Universal Common Ancestor ◽

Trna Synthetases ◽

Universal Common Ancestor

The twenty amino acids in the standard genetic code were fixed prior to the last universal common ancestor (LUCA). Factors that guided this selection included establishment of pathways for their metabolic synthesis and the concomitant fixation of substrate specificities in the emerging aminoacyl-tRNA synthetases (aaRSs). In this conceptual paper, we propose that the chemical reactivity of some amino acid side chains (e.g., lysine, cysteine, homocysteine, ornithine, homoserine, and selenocysteine) delayed or prohibited the emergence of the corresponding aaRSs and helped define the amino acids in the standard genetic code. We also consider the possibility that amino acid chemistry delayed the emergence of the glutaminyl- and asparaginyl-tRNA synthetases, neither of which are ubiquitous in extant organisms. We argue that fundamental chemical principles played critical roles in fixation of some aspects of the genetic code pre- and post-LUCA.

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Mutations activating the yeast eIF-2 alpha kinase GCN2: isolation of alleles altering the domain related to histidyl-tRNA synthetases

Molecular and Cellular Biology ◽

10.1128/mcb.12.12.5801-5815.1992 ◽

1992 ◽

Vol 12 (12) ◽

pp. 5801-5815

Author(s):

M Ramirez ◽

R C Wek ◽

C R Vazquez de Aldana ◽

B M Jackson ◽

B Freeman ◽

...

Keyword(s):

Amino Acid ◽

Protein Kinase ◽

Translation Initiation ◽

Trna Synthetase ◽

Amino Acid Starvation ◽

Translation Initiation Factor ◽

Initiation Factor ◽

Atp Binding ◽

Sequence Motif ◽

Trna Synthetases

The protein kinase GCN2 stimulates expression of the yeast transcriptional activator GCN4 at the translational level by phosphorylating the alpha subunit of translation initiation factor 2 (eIF-2 alpha) in amino acid-starved cells. Phosphorylation of eIF-2 alpha reduces its activity, allowing ribosomes to bypass short open reading frames present in the GCN4 mRNA leader and initiate translation at the GCN4 start codon. We describe here 17 dominant GCN2 mutations that lead to derepression of GCN4 expression in the absence of amino acid starvation. Seven of these GCN2c alleles map in the protein kinase moiety, and two in this group alter the presumed ATP-binding domain, suggesting that ATP binding is a regulated aspect of GCN2 function. Six GCN2c alleles map in a region related to histidyl-tRNA synthetases, and two in this group alter a sequence motif conserved among class II aminoacyl-tRNA synthetases that directly interacts with the acceptor stem of tRNA. These results support the idea that GCN2 kinase function is activated under starvation conditions by binding uncharged tRNA to the domain related to histidyl-tRNA synthetase. The remaining GCN2c alleles map at the extreme C terminus, a domain required for ribosome association of the protein. Representative mutations in each domain were shown to depend on the phosphorylation site in eIF-2 alpha for their effects on GCN4 expression and to increase the level of eIF-2 alpha phosphorylation in the absence of amino acid starvation. Synthetic GCN2c double mutations show greater derepression of GCN4 expression than the parental single mutations, and they have a slow-growth phenotype that we attribute to inhibition of general translation initiation. The phenotypes of the GCN2c alleles are dependent on GCN1 and GCN3, indicating that these two positive regulators of GCN4 expression mediate the inhibitory effects on translation initiation associated with activation of the yeast eIF-2 alpha kinase GCN2.

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relA-Independent Amino Acid Starvation Response Network of Streptococcus pyogenes

Journal of Bacteriology ◽

10.1128/jb.183.24.7354-7364.2001 ◽

2001 ◽

Vol 183 (24) ◽

pp. 7354-7364 ◽

Cited By ~ 52

Author(s):

Kerstin Steiner ◽

Horst Malke

Keyword(s):

Amino Acid ◽

Streptococcus Pyogenes ◽

Target Genes ◽

Group A Streptococcus ◽

Housekeeping Genes ◽

Trna Synthetase ◽

Amino Acid Starvation ◽

Infection Process ◽

Starvation Response ◽

Virulence Gene Expression

ABSTRACT Streptococcus pyogenes (group A streptococcus [GAS]), a multiple-amino-acid-auxotrophic human pathogen, may face starvation for essential amino acids during various stages of the infection process. Since the response of GAS to such conditions is likely to influence pathogenetic processes, we set out to identify by transcriptional analyses genes and operons that are responsive to amino acid starvation and examined whether functionally meaningful response patterns can be ascertained. We discovered that GAS are capable of mounting a relA-independent amino acid starvation response that involves transcriptional modulation of a wide array of housekeeping genes as well as accessory and dedicated virulence genes. Housekeeping genes that were upregulated during starvation of both wild-type and relA mutant strains included the newly identified T-box members of the aminoacyl-tRNA synthetase genes, the genes for components of the tmRNA-mediated peptide tagging and proteolysis system for abnormal proteins (ssrA, smpB,clpP, and clpC), and the operons for thednaK and groE groups of molecular chaperones. In addition to upregulation of the genes for oligopeptide permease (opp), intracellular peptidase (pepB), and the two-component regulatorcovRS reported previously (K. Steiner and H. Malke, Mol. Microbiol. 38:1004–1016, 2000), amino acid starvation stimulated the transcription of the growth phase-associated, virulence-regulatory fas operon, the streptolysin S operon (sag), and the gene for autoinducer-2 production protein (luxS). A prominent feature of operons exhibiting internal transcriptional termination (opp, fas, andsag) was starvation-promoted full-length transcription, a mechanism that improves the efficacy of these systems by increasing the level of coordinate transcription of functionally related genes. Based on these results, a regulatory network with feedback mechanisms is proposed that counteracts the stringent response, links the levels of key rate-limiting enzymes to virulence gene expression, and enables the organism in a dynamic way to take advantage of protein-rich environments provided by its human host. As several of the affected target genes are controlled by more than one regulator, fine modulation may result in accordance with the demands imposed by ecologically different colonization sites upon the adaptive capacity of the pathogen.

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Cross-editing by a tRNA synthetase allows vertebrates to abundantly express mischargeable tRNA without causing mistranslation

Nucleic Acids Research ◽

10.1093/nar/gkaa469 ◽

2020 ◽

Vol 48 (12) ◽

pp. 6445-6457 ◽

Cited By ~ 4

Author(s):

Meirong Chen ◽

Bernhard Kuhle ◽

Jolene Diedrich ◽

Ze Liu ◽

James J Moresco ◽

...

Keyword(s):

Amino Acids ◽

Amino Acid ◽

Mammalian Cells ◽

Trna Synthetase ◽

Intrinsic Activity ◽

Trna Synthetases ◽

Evolutionarily Conserved ◽

Trna Species ◽

In Trans ◽

Editing Activity

Abstract The accuracy in pairing tRNAs with correct amino acids by aminoacyl-tRNA synthetases (aaRSs) dictates the fidelity of translation. To ensure fidelity, multiple aaRSs developed editing functions that remove a wrong amino acid from tRNA before it reaches the ribosome. However, no specific mechanism within an aaRS is known to handle the scenario where a cognate amino acid is mischarged onto a wrong tRNA, as exemplified by AlaRS mischarging alanine to G4:U69-containing tRNAThr. Here, we report that the mischargeable G4:U69-containing tRNAThr are strictly conserved in vertebrates and are ubiquitously and abundantly expressed in mammalian cells and tissues. Although these tRNAs are efficiently mischarged, no corresponding Thr-to-Ala mistranslation is detectable. Mistranslation is prevented by a robust proofreading activity of ThrRS towards Ala-tRNAThr. Therefore, while wrong amino acids are corrected within an aaRS, a wrong tRNA is handled in trans by an aaRS cognate to the mischarged tRNA species. Interestingly, although Ala-tRNAThr mischarging is not known to occur in bacteria, Escherichia coli ThrRS also possesses robust cross-editing ability. We propose that the cross-editing activity of ThrRS is evolutionarily conserved and that this intrinsic activity allows G4:U69-containing tRNAThr to emerge and be preserved in vertebrates to have alternative functions without compromising translational fidelity.

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Mutations activating the yeast eIF-2 alpha kinase GCN2: isolation of alleles altering the domain related to histidyl-tRNA synthetases.

Molecular and Cellular Biology ◽

10.1128/mcb.12.12.5801 ◽

1992 ◽

Vol 12 (12) ◽

pp. 5801-5815 ◽

Cited By ~ 77

Author(s):

M Ramirez ◽

R C Wek ◽

C R Vazquez de Aldana ◽

B M Jackson ◽

B Freeman ◽

...

Keyword(s):

Amino Acid ◽

Protein Kinase ◽

Translation Initiation ◽

Trna Synthetase ◽

Amino Acid Starvation ◽

Translation Initiation Factor ◽

Initiation Factor ◽

Atp Binding ◽

Sequence Motif ◽

Trna Synthetases

The protein kinase GCN2 stimulates expression of the yeast transcriptional activator GCN4 at the translational level by phosphorylating the alpha subunit of translation initiation factor 2 (eIF-2 alpha) in amino acid-starved cells. Phosphorylation of eIF-2 alpha reduces its activity, allowing ribosomes to bypass short open reading frames present in the GCN4 mRNA leader and initiate translation at the GCN4 start codon. We describe here 17 dominant GCN2 mutations that lead to derepression of GCN4 expression in the absence of amino acid starvation. Seven of these GCN2c alleles map in the protein kinase moiety, and two in this group alter the presumed ATP-binding domain, suggesting that ATP binding is a regulated aspect of GCN2 function. Six GCN2c alleles map in a region related to histidyl-tRNA synthetases, and two in this group alter a sequence motif conserved among class II aminoacyl-tRNA synthetases that directly interacts with the acceptor stem of tRNA. These results support the idea that GCN2 kinase function is activated under starvation conditions by binding uncharged tRNA to the domain related to histidyl-tRNA synthetase. The remaining GCN2c alleles map at the extreme C terminus, a domain required for ribosome association of the protein. Representative mutations in each domain were shown to depend on the phosphorylation site in eIF-2 alpha for their effects on GCN4 expression and to increase the level of eIF-2 alpha phosphorylation in the absence of amino acid starvation. Synthetic GCN2c double mutations show greater derepression of GCN4 expression than the parental single mutations, and they have a slow-growth phenotype that we attribute to inhibition of general translation initiation. The phenotypes of the GCN2c alleles are dependent on GCN1 and GCN3, indicating that these two positive regulators of GCN4 expression mediate the inhibitory effects on translation initiation associated with activation of the yeast eIF-2 alpha kinase GCN2.

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Oxidation of cellular amino acid pools leads to cytotoxic mistranslation of the genetic code

eLife ◽

10.7554/elife.02501 ◽

2014 ◽

Vol 3 ◽

Cited By ~ 56

Author(s):

Tammy J Bullwinkle ◽

Noah M Reynolds ◽

Medha Raina ◽

Adil Moghal ◽

Eleftheria Matsa ◽

...

Keyword(s):

Amino Acids ◽

Protein Synthesis ◽

Amino Acid ◽

Genetic Code ◽

Trna Synthetase ◽

Control Mechanisms ◽

Cellular Toxicity ◽

Trna Synthetases ◽

Aminoacyl Trna Synthetases ◽

The Cost

Aminoacyl-tRNA synthetases use a variety of mechanisms to ensure fidelity of the genetic code and ultimately select the correct amino acids to be used in protein synthesis. The physiological necessity of these quality control mechanisms in different environments remains unclear, as the cost vs benefit of accurate protein synthesis is difficult to predict. We show that in Escherichia coli, a non-coded amino acid produced through oxidative damage is a significant threat to the accuracy of protein synthesis and must be cleared by phenylalanine-tRNA synthetase in order to prevent cellular toxicity caused by mis-synthesized proteins. These findings demonstrate how stress can lead to the accumulation of non-canonical amino acids that must be excluded from the proteome in order to maintain cellular viability.

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Fine-Tuning of Alanyl-tRNA Synthetase Quality Control Alleviates Global Dysregulation of the Proteome

Genes ◽

10.3390/genes11101222 ◽

2020 ◽

Vol 11 (10) ◽

pp. 1222

Author(s):

Paul Kelly ◽

Arundhati Kavoor ◽

Michael Ibba

Keyword(s):

Amino Acids ◽

Quality Control ◽

Amino Acid ◽

Antibiotic Sensitivity ◽

Trna Synthetase ◽

Fine Tuning ◽

Trna Synthetases ◽

E Coli ◽

Suppressor Mutations ◽

Cognate Trna

One integral step in the transition from a nucleic acid encoded-genome to functional proteins is the aminoacylation of tRNA molecules. To perform this activity, aminoacyl-tRNA synthetases (aaRSs) activate free amino acids in the cell forming an aminoacyl-adenylate before transferring the amino acid on to its cognate tRNA. These newly formed aminoacyl-tRNA (aa-tRNA) can then be used by the ribosome during mRNA decoding. In Escherichia coli, there are twenty aaRSs encoded in the genome, each of which corresponds to one of the twenty proteinogenic amino acids used in translation. Given the shared chemicophysical properties of many amino acids, aaRSs have evolved mechanisms to prevent erroneous aa-tRNA formation with non-cognate amino acid substrates. Of particular interest is the post-transfer proofreading activity of alanyl-tRNA synthetase (AlaRS) which prevents the accumulation of Ser-tRNAAla and Gly-tRNAAla in the cell. We have previously shown that defects in AlaRS proofreading of Ser-tRNAAla lead to global dysregulation of the E. coli proteome, subsequently causing defects in growth, motility, and antibiotic sensitivity. Here we report second-site AlaRS suppressor mutations that alleviate the aforementioned phenotypes, revealing previously uncharacterized residues within the AlaRS proofreading domain that function in quality control.

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Evolution of Life on Earth: tRNA, Aminoacyl-tRNA Synthetases and the Genetic Code

Life ◽

10.3390/life10030021 ◽

2020 ◽

Vol 10 (3) ◽

pp. 21 ◽

Cited By ~ 6

Author(s):

Lei Lei ◽

Zachary F Burton

Keyword(s):

Amino Acids ◽

Amino Acid ◽

Genetic Code ◽

Elongation Factor ◽

Trna Synthetase ◽

Aminoacyl Trna Synthetase ◽

Trna Synthetases ◽

Amino Acid Code ◽

Evolution Of Life ◽

Life On Earth

Life on Earth and the genetic code evolved around tRNA and the tRNA anticodon. We posit that the genetic code initially evolved to synthesize polyglycine as a cross-linking agent to stabilize protocells. We posit that the initial amino acids to enter the code occupied larger sectors of the code that were then invaded by incoming amino acids. Displacements of amino acids follow selection rules. The code sectored from a glycine code to a four amino acid code to an eight amino acid code to an ~16 amino acid code to the standard 20 amino acid code with stops. The proposed patterns of code sectoring are now most apparent from patterns of aminoacyl-tRNA synthetase evolution. The Elongation Factor-Tu GTPase anticodon-codon latch that checks the accuracy of translation appears to have evolved at about the eight amino acid to ~16 amino acid stage. Before evolution of the EF-Tu latch, we posit that both the 1st and 3rd anticodon positions were wobble positions. The genetic code evolved via tRNA charging errors and via enzymatic modifications of amino acids joined to tRNAs, followed by tRNA and aminoacyl-tRNA synthetase differentiation. Fidelity mechanisms froze the code by inhibiting further innovation.

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