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

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

Nucleic Acids Research ◽

10.1093/nar/gkaa055 ◽

2020 ◽

Vol 48 (6) ◽

pp. 3071-3088

Author(s):

Matthew R McFarland ◽

Corina D Keller ◽

Brandon M Childers ◽

Stephen A Adeniyi ◽

Holly Corrigall ◽

...

Keyword(s):

Amino Acids ◽

Amino Acid ◽

Neurological Disorders ◽

Trna Synthetase ◽

Northern Blotting ◽

Amino Acid Starvation ◽

Starvation Response ◽

Kinase Activation ◽

Trna Synthetases ◽

Starvation Conditions

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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Quality control in tRNA charging -- editing of homocysteine.

Acta Biochimica Polonica ◽

10.18388/abp.2011_2259 ◽

2011 ◽

Vol 58 (2) ◽

Cited By ~ 34

Author(s):

Hieronim Jakubowski

Keyword(s):

Amino Acids ◽

Quality Control ◽

Control Point ◽

Building Blocks ◽

Trna Synthetase ◽

Nutritional Deficiencies ◽

Homocysteine Thiolactone ◽

Trna Synthetases ◽

Aminoacyl Trna Synthetases ◽

Living Organisms

All living organisms conduct protein synthesis with a high degree of accuracy maintained in the transmission and flow of information from a gene to protein product. One crucial 'quality control' point in maintaining a high level of accuracy is the selectivity by which aminoacyl-tRNA synthetases furnish correctly activated amino acids, attached to tRNA species, as the building blocks for growing protein chains. When differences in binding energies of amino acids to an aminoacyl-tRNA synthetase are inadequate, editing is used as a major determinant of enzyme selectivity. Some incorrect amino acids are edited at the active site before the transfer to tRNA (pre-transfer editing), while others are edited after transfer to tRNA at a separate editing site (post-transfer editing). Access of natural non-protein amino acids, such as homocysteine, homoserine, or ornithine to the genetic code is prevented by the editing function of aminoacyl-tRNA synthetases. Disabling editing function leads to tRNA mischarging errors and incorporation of incorrect amino acids into protein, which is detrimental to cell homeostasis and inhibits growth. Continuous homocysteine editing by methionyl-tRNA synthetase, resulting in the synthesis of homocysteine thiolactone, is part of the process of tRNA aminoacylation in living organisms, from bacteria to man. Excessive homocysteine thiolactone synthesis in hyperhomocysteinemia caused by genetic or nutritional deficiencies is linked to human vascular and neurological diseases.

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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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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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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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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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Free-energy dissipation constrainst on the accuracy of enzymatic selections

Quarterly Reviews of Biophysics ◽

10.1017/s0033583500001669 ◽

1980 ◽

Vol 13 (2) ◽

pp. 231-254 ◽

Cited By ~ 23

Author(s):

C. Blomberg ◽

M. Ehrenberg ◽

C. G. Kurland

Keyword(s):

Amino Acids ◽

Free Energy ◽

Protein Synthesis ◽

Amino Acid ◽

Trna Synthetases ◽

Aminoacyl Trna Synthetases ◽

Error Frequency ◽

Structural Differences ◽

Cognate Trna ◽

Selection Of

A bacterium incorporates amino acids into protein with an error frequency close to one in three thousand (Edelman & Gallant, 1977). Nevertheless, the structural differences between related amino acids are so small that it is difficult to see how they can be distinguished from each other with such accuracy (see, for example, Pauling, 1958).Indeed, the selection of amino acids during protein synthesis is carried out twice: first by the aminoacyl-tRNA synthetases and then by the codon-programmed ribosome. Each of these substrate selections is in fact a double selection. In the case of the synthetase both a particular amino acid and a corresponding cognate tRNA must be chosen to form the aminoacyl-tRNA. On the ribosome, the aminoacyl-tRNA must be matched with a cognate codon, and then the mRNA must be advanced by exactly one codon length to position the next codon in the appropriate ribosome site so that it too can be translated.

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

10.1101/610790 ◽

2019 ◽

Cited By ~ 2

Author(s):

Matthew R. McFarland ◽

Corina D. Keller ◽

Brandon M. Childers ◽

Stephen A. Adeniyi ◽

Holly Corrigall ◽

...

Keyword(s):

Amino Acids ◽

Amino Acid ◽

Neurological Disorders ◽

Trna Synthetase ◽

Northern Blotting ◽

Amino Acid Starvation ◽

Starvation Response ◽

Kinase Activation ◽

Trna Synthetases ◽

Starvation Conditions

ABSTRACTDuring 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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Relaxed Substrate Specificity Leads to Extensive tRNA Mischarging by Streptococcus pneumoniae Class I and Class II Aminoacyl-tRNA Synthetases

mBio ◽

10.1128/mbio.01656-14 ◽

2014 ◽

Vol 5 (5) ◽

Cited By ~ 8

Author(s):

Jennifer Shepherd ◽

Michael Ibba

Keyword(s):

Quality Control ◽

Cell Wall ◽

Protein Synthesis ◽

Streptococcus Pneumoniae ◽

Amino Acid ◽

Substrate Specificity ◽

Trna Synthetase ◽

Content Type ◽

Trna Synthetases ◽

Aminoacyl Trna Synthetases

ABSTRACTAminoacyl-tRNA synthetases provide the first step in protein synthesis quality control by discriminating cognate from noncognate amino acid and tRNA substrates. While substrate specificity is enhanced in many instances bycis-andtrans-editing pathways, it has been revealed that in organisms such asStreptococcus pneumoniaesome aminoacyl-tRNA synthetases display significant tRNA mischarging activity. To investigate the extent of tRNA mischarging in this pathogen, the aminoacylation profiles of class I isoleucyl-tRNA synthetase (IleRS) and class II lysyl-tRNA synthetase (LysRS) were determined. Pneumococcal IleRS mischarged tRNAIlewith both Val, as demonstrated in other bacteria, and Leu in a tRNA sequence-dependent manner. IleRS substrate specificity was achieved in an editing-independent manner, indicating that tRNA mischarging would only be significant under growth conditions where Ile is depleted. Pneumococcal LysRS was found to misaminoacylate tRNALyswith Ala and to a lesser extent Thr and Ser, with mischarging efficiency modulated by the presence of an unusual U4:G69 wobble pair in the acceptor stems of both pneumococcal tRNALysisoacceptors. Addition of thetrans-editing factor MurM, which also functions in peptidoglycan synthesis, reduced Ala-tRNALysproduction by LysRS, providing evidence for cross talk between the protein synthesis and cell wall biogenesis pathways. Mischarging of tRNALysby AlaRS was also observed, and this would provide additional potential MurM substrates. More broadly, the extensive mischarging activities now described for a number ofStreptococcus pneumoniaeaminoacyl-tRNA synthetases suggest that adaptive misaminoacylation may contribute significantly to the viability of this pathogen during amino acid starvation.IMPORTANCEStreptococcus pneumoniaeis a common causative agent of several debilitating and potentially life-threatening infections, such as pneumonia, meningitis, and infectious endocarditis. Such infections are increasingly difficult to treat due to widespread development of penicillin resistance. High-level penicillin resistance is known to depend in part upon MurM, a protein involved in both aminoacyl-tRNA-dependent synthesis of indirect amino acid cross-linkages within cell wall peptidoglycan and in translation quality control. The involvement of MurM in both protein synthesis and antibiotic resistance identify it as a potential target for the development of new and potent antibiotics for pneumococcal infections. The goals of this work were to identify and characterizeS. pneumoniaepathways that can synthesize mischarged tRNAs and to relate these activities to expected changes in protein and peptidoglycan biosynthesis during antibiotic and nutritional stress.

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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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