GO:0004813 alanine-tRNA ligase activity: Mechanism, Genes and Research Methods
Research-grade guide for scientists and biopharma professionals
Key Takeaways
• GO:0004813 (alanine-tRNA ligase activity) is the molecular function that attaches L-alanine to tRNA(Ala), a critical step in protein synthesis.
• The reaction consumes ATP and L-alanine to produce L-alanyl-tRNA(Ala), AMP, and diphosphate, as defined by QuickGO.
• AlaRS, encoded by AARS1 in humans, is the enzyme responsible for this activity and is highly conserved from bacteria to humans.
• Mutations in AARS1 cause Charcot-Marie-Tooth disease and other neurological disorders, making it a disease-relevant target [3,7].
• AARS1 has moonlighting functions, including acting as a lactate sensor and lactyltransferase that modifies p53 and YAP, linking it to cancer [1,2].
• CRISPR knockout, point mutation, and knock-in models are essential for dissecting the role of alanine-tRNA ligase activity in health and disease.
Description
Alanine-tRNA ligase activity (GO:0004813) is a fundamental molecular function that ensures the correct incorporation of alanine into proteins during translation. This activity is catalyzed by alanyl-tRNA synthetase (AlaRS), an enzyme that charges tRNA(Ala) with L-alanine in a two-step reaction [4,7]. The fidelity of this process is critical for maintaining proteome integrity, as errors can lead to misfolded proteins and cellular stress. Researchers study this term to understand basic translation mechanisms, tRNA synthetase evolution, and the growing list of human diseases linked to mutations in AARS1, the gene encoding the human AlaRS [3,7]. Beyond its canonical role, AlaRS has been implicated in cancer through non-canonical functions such as lactylation of p53 and YAP [1,2]. Thus, GO:0004813 represents both a core housekeeping function and a node for disease-relevant signaling.
alanine-tRNA ligase activity At A Glance
| GO ID | GO:0004813 |
|---|---|
| GO term | alanine-tRNA ligase activity |
| Ontology | molecular_function |
| Synonym | alanyl-tRNA synthetase activity; AlaRS; alanine translase activity |
| Major function | Catalyzes the attachment of L-alanine to tRNA(Ala) for protein synthesis |
| Reaction | ATP + L-alanine + tRNA(Ala) = AMP + diphosphate + L-alanyl-tRNA(Ala) |
| Enzyme class | Ligase (aminoacyl-tRNA synthetase, class II) |
| Human gene | AARS1 |
| Disease relevance | Charcot-Marie-Tooth disease, cancer, neurological disorders |
What Is GO:0004813?
Alanine-tRNA ligase activity is the catalysis of the reaction: ATP + L-alanine + tRNA(Ala) = AMP + diphosphate + L-alanyl-tRNA(Ala). This definition, from QuickGO, describes the covalent attachment of the amino acid alanine to its cognate transfer RNA, a prerequisite for decoding the alanine codon during protein synthesis.
Why Is alanine-tRNA ligase activity Important in Cell Biology?
Alanine-tRNA ligase activity is essential for translating the genetic code into functional proteins, as it ensures that alanine is correctly paired with its tRNA. Disruption of this activity leads to global defects in protein synthesis and triggers cellular stress responses. In humans, mutations in AARS1 cause Charcot-Marie-Tooth disease, a peripheral neuropathy, highlighting the clinical importance of this function. Moreover, recent studies have revealed that AARS1 can act as a lactyltransferase, modifying proteins like p53 and YAP, which links alanine-tRNA ligase activity to tumorigenesis and metabolic signaling [1,2]. Therefore, understanding GO:0004813 is crucial for both fundamental biology and therapeutic development.
• Ensures accurate translation of alanine codons, preventing proteotoxic stress.
• Mutations in AARS1 cause Charcot-Marie-Tooth disease and other neuropathies.
• AARS1 is a lactate sensor and lactyltransferase that modifies p53, contributing to tumorigenesis.
• AARS1 promotes YAP signaling in gastric cancer through its lactyltransferase activity.
• AlaRS is a target for antibiotic development due to its essential role in bacteria.
• Studying AlaRS provides insights into tRNA synthetase evolution and aminoacylation fidelity.
• Alanine-tRNA ligase activity is required for mitochondrial translation and cellular respiration.
• Dysregulation of AARS1 is linked to metabolic reprogramming in cancer [1,2].
• Aptamer-tagged tRNAs can be used to study AlaRS activity and tRNA charging in vitro.
• Animal models of AARS1 mutations recapitulate neuropathy phenotypes, aiding drug discovery.
Mechanism, Genes and Research Methods
What Happens During alanine-tRNA ligase activity?
In simple terms: The enzyme grabs alanine and attaches it to a specific tRNA molecule, using energy from ATP.
Alanine-tRNA ligase activity proceeds through a two-step aminoacylation reaction. First, L-alanine is activated by ATP to form an alanyl-adenylate intermediate, releasing pyrophosphate. Second, the alanyl group is transferred to the 3' end of tRNA(Ala), yielding L-alanyl-tRNA(Ala) and AMP [4,7]. This mechanism ensures high fidelity, as the enzyme discriminates against similar amino acids like glycine and serine. The reaction is essential for decoding the alanine codon during translation.
Structural Basis of Substrate Recognition
In simple terms: The enzyme has a pocket that fits alanine perfectly, and a separate domain that recognizes the tRNA's shape.
AlaRS belongs to the class II aminoacyl-tRNA synthetases and functions as a homodimer or homotetramer depending on the organism. The catalytic domain contains the active site for alanine activation, while the tRNA-binding domain recognizes the acceptor stem and anticodon loop of tRNA(Ala). In Escherichia coli AlaRS, a conserved editing domain hydrolyzes mischarged tRNA species to maintain fidelity. Structural studies have revealed conformational changes upon substrate binding that facilitate catalysis.
Molecular Mechanism and Cofactors
In simple terms: The enzyme uses ATP as an energy source and requires magnesium ions to work.
The catalytic mechanism of alanine-tRNA ligase activity requires ATP and Mg2+ as cofactors. The reaction follows an ordered sequential mechanism where ATP and alanine bind first, followed by tRNA. The enzyme catalyzes the formation of an aminoacyl-adenylate intermediate, which then reacts with the 2'-OH of the terminal adenosine of tRNA(Ala). Editing activity hydrolyzes misactivated amino acids, ensuring only alanine is incorporated.
Regulation of Alanine-tRNA Ligase Activity
In simple terms: The cell controls how much of this enzyme is made and how active it is, depending on its needs.
Alanine-tRNA ligase activity is regulated at multiple levels. Transcription of AARS1 can be induced by stress and growth signals. Post-translational modifications, such as phosphorylation, may modulate enzyme activity. Additionally, AARS1 can sense lactate levels and act as a lactyltransferase, linking metabolic state to its non-canonical functions [1,2]. This moonlighting activity is independent of its aminoacylation function and contributes to cancer progression [1,2].
Key Genes Involved in GO:0004813 alanine-tRNA ligase activity
The following genes and proteins are directly involved in or regulate alanine-tRNA ligase activity and its associated pathways.
| Gene | Major Role | Research Relevance |
|---|---|---|
| AARS1 | Encodes human alanyl-tRNA synthetase; catalyzes alanine-tRNA ligation | Mutations cause Charcot-Marie-Tooth disease; lactyltransferase in cancer [1,2,3,7] |
| AARS2 | Mitochondrial alanyl-tRNA synthetase | Mutations cause mitochondrial disorders; studied for organelle translation |
| alaS (E. coli) | Bacterial alanyl-tRNA synthetase | Model for substrate recognition and antibiotic targeting |
| tRNA(Ala) | Transfer RNA specific for alanine | Substrate for aminoacylation; studied with aptamer tags |
| p53 | Tumor suppressor; target of AARS1 lactylation | Lactylation by AARS1 affects p53 function in tumorigenesis |
| YAP | Transcriptional co-activator; target of AARS1 lactylation | AARS1 promotes YAP signaling in gastric cancer |
| GDF15 | Stress-induced cytokine; biomarker in CMT | Elevated in AARS1-related neuropathy |
| NCAM1 | Neural cell adhesion molecule; biomarker in CMT | Altered in Charcot-Marie-Tooth disease |
| TyrRS | Tyrosyl-tRNA synthetase; related synthetase | Evolutionary comparison of aminoacylation systems |
| GlyRS | Glycyl-tRNA synthetase; related synthetase | Comparative studies of synthetase mechanisms |
| ATP | Energy source for aminoacylation | Required cofactor for the reaction |
| L-alanine | Substrate amino acid | Directly incorporated into tRNA(Ala) |
| AMP | Reaction product | Byproduct of aminoacylation |
| Diphosphate | Reaction product | Released during activation step |
| L-alanyl-tRNA(Ala) | Product; delivers alanine to ribosome | Essential for protein synthesis |
| AlaRS editing domain | Hydrolyzes mischarged tRNA | Maintains translational fidelity |
| Lactate | Metabolite sensed by AARS1 | Regulates lactyltransferase activity |
| mTOR pathway | Regulates protein synthesis | May influence AlaRS expression |
How Is alanine-tRNA ligase activity Regulated?
Alanine-tRNA ligase activity is regulated at transcriptional, post-transcriptional, and post-translational levels. The expression of AARS1 can be induced by cellular stress and growth factors, and its activity may be modulated by phosphorylation. Additionally, AARS1 functions as a lactate sensor, where elevated lactate levels promote its lactyltransferase activity, leading to lactylation of p53 and YAP, which affects tumorigenesis and signaling [1,2]. This non-canonical regulation links metabolic status to alanine-tRNA ligase function.
alanine-tRNA ligase activity and Human Disease
| Gene | Disease / Biology | Potential Experimental Model |
|---|---|---|
| AARS1 | Charcot-Marie-Tooth disease | Knock-in mouse models with patient mutations [3,7] |
| AARS1 | Cancer (gastric, others) | Xenograft models with AARS1 knockout or overexpression [1,2] |
| AARS2 | Mitochondrial cardiomyopathy | Patient-derived fibroblasts and iPSC-derived cardiomyocytes |
| p53 | Tumor suppression | Cell lines with p53 lactylation site mutations |
| YAP | Gastric cancer progression | Organoids and mouse models with YAP lactylation mutants |
Charcot-Marie-Tooth Disease and Neurodegeneration
Mutations in AARS1 cause Charcot-Marie-Tooth disease, a hereditary peripheral neuropathy characterized by progressive muscle weakness and sensory loss [3,7]. These mutations often impair aminoacylation activity, leading to protein synthesis defects in neurons. Biomarkers such as NCAM1 and GDF15 are elevated in patients and mouse models, providing tools for diagnosis and monitoring. The disease highlights the critical role of alanine-tRNA ligase activity in neuronal health.
Cancer and Metabolic Reprogramming
AARS1 is overexpressed in various cancers and contributes to tumorigenesis through non-canonical functions. It acts as a lactyltransferase that lactylates p53, inhibiting its tumor suppressor activity. In gastric cancer, AARS1 lactylates YAP, promoting its transcriptional activity and cancer progression. These findings link alanine-tRNA ligase activity to metabolic reprogramming and offer potential therapeutic targets.
Mitochondrial Disorders
The mitochondrial alanyl-tRNA synthetase, AARS2, is essential for mitochondrial translation. Mutations in AARS2 cause infantile mitochondrial cardiomyopathy and other disorders. Studying alanine-tRNA ligase activity in mitochondria provides insights into organelle-specific translation and disease mechanisms.
From alanine-tRNA ligase activity-Related Genes to Experimental Models
| Research Question | Suitable Model |
|---|---|
| What is the effect of AARS1 loss on translation? | CRISPR knockout in HEK293 or HeLa cells |
| How do disease mutations affect aminoacylation? | Point mutation knock-in in patient fibroblasts |
| Does AARS1 lactylation of p53 require its synthetase activity? | Knock-in of catalytically dead AARS1 |
| Can AARS1 be targeted for cancer therapy? | Overexpression and knockout in cancer cell lines |
| What is the role of mitochondrial AlaRS? | AARS2 knockout in mitochondrial reporter cells |
| How is tRNA(Ala) recognition achieved? | In vitro aminoacylation assays with mutant tRNAs |
How to Study the alanine-tRNA ligase activity Process
| Method | What It Measures | Typical Application |
|---|---|---|
| Aminoacylation assay | tRNA charging activity | Enzyme kinetics and inhibitor testing |
| Ribo-seq | Translation efficiency and codon usage | Global effects of AARS1 loss |
| RNA-seq | Transcriptome changes | Stress response and gene expression |
| Proteomics | Protein abundance and modifications | Lactylome analysis [1,2] |
| CRISPR screen | Gene essentiality and interactions | Identifying synthetic lethality |
| Western blot | Protein expression and lactylation | Validating AARS1 targets |
| Immunofluorescence | Subcellular localization | Mitochondrial vs cytoplasmic AlaRS |
| qPCR | mRNA levels | AARS1 expression in disease models |
Aminoacylation Assays
In vitro aminoacylation assays measure the incorporation of radiolabeled alanine into tRNA(Ala) using purified AlaRS. These assays are used to determine kinetic parameters and fidelity. They can be adapted for high-throughput screening of inhibitors.
Ribo-seq and RNA-seq
Ribosome profiling (Ribo-seq) measures global translation efficiency and can reveal defects in alanine codon decoding upon AARS1 perturbation. RNA-seq provides transcriptomic changes, including stress response genes.
Proteomics and Lactylome Analysis
Mass spectrometry-based proteomics can identify lactylated proteins modified by AARS1, such as p53 and YAP [1,2]. Lactylome profiling reveals the broader impact of AARS1 lactyltransferase activity on cellular signaling.
CRISPR Screens and Functional Genomics
Genome-wide CRISPR knockout screens can identify synthetic lethal interactions with AARS1 mutations or identify modifiers of alanine-tRNA ligase activity. These screens are powerful for discovering new therapeutic targets.
How CRISPR Can Be Used to Study GO:0004813 alanine-tRNA ligase activity
Knockout
CRISPR knockout of AARS1 in cell lines abolishes alanine-tRNA ligase activity, leading to translation arrest and cell death. Knockout models are used to study the essentiality of this function and to identify compensatory pathways.
Point Mutation
Point mutations in AARS1 that mimic patient alleles can be introduced via CRISPR to study disease mechanisms. For example, mutations in the editing domain can cause misfolding and neurodegeneration.
Knock-in
Knock-in of tagged AARS1 (e.g., FLAG or GFP) allows for localization and interaction studies. Knock-in of lactylation-deficient mutants can dissect non-canonical functions.
Overexpression
CRISPR activation or lentiviral overexpression of AARS1 can model cancers with elevated alanine-tRNA ligase activity [1,2]. Overexpression studies reveal oncogenic roles and metabolic rewiring.
How EDITGENE Supports alanine-tRNA ligase activity Research
Researchers studying alanine-tRNA ligase activity-related genes often need to determine whether a candidate gene is causally involved in translation fidelity, disease, or cancer. EDITGENE provides a comprehensive suite of CRISPR services to accelerate this research.
Contact EDITGENE today to design your custom CRISPR model for alanine-tRNA ligase activity research.
Frequently Asked Questions About alanine-tRNA ligase activity
What is alanine-tRNA ligase activity?
Alanine-tRNA ligase activity (GO:0004813) is the molecular function that attaches the amino acid alanine to its corresponding transfer RNA, a key step in protein synthesis.
What genes are involved in alanine-tRNA ligase activity?
The primary gene is AARS1 in humans, which encodes alanyl-tRNA synthetase. AARS2 encodes the mitochondrial version.
What diseases are associated with alanine-tRNA ligase activity?
Mutations in AARS1 cause Charcot-Marie-Tooth disease, and the enzyme is implicated in cancer through lactylation of p53 and YAP [1,2,3].
How is alanine-tRNA ligase activity regulated?
It is regulated by transcription, post-translational modifications, and metabolic signals such as lactate levels [1,7].
What is the reaction catalyzed by alanine-tRNA ligase?
The reaction is: ATP + L-alanine + tRNA(Ala) = AMP + diphosphate + L-alanyl-tRNA(Ala).
Can CRISPR be used to study alanine-tRNA ligase activity?
Yes, CRISPR knockout, point mutation, and knock-in models are widely used to study AARS1 function and disease mutations.
What are the symptoms of Charcot-Marie-Tooth disease linked to AARS1?
Symptoms include progressive muscle weakness, sensory loss, and reduced nerve conduction velocities.
Is alanine-tRNA ligase a target for antibiotics?
Bacterial AlaRS is essential and differs from human AlaRS, making it a potential antibiotic target.
What is the role of AARS1 in cancer?
AARS1 acts as a lactyltransferase that modifies p53 and YAP, promoting tumorigenesis and cancer progression [1,2].
How can I model alanine-tRNA ligase mutations in the lab?
You can use CRISPR to introduce patient-specific mutations into AARS1 in cell lines or animal models [3,7].
Conclusion
Alanine-tRNA ligase activity (GO:0004813) is a cornerstone of protein synthesis, ensuring the accurate incorporation of alanine into proteins. Its importance extends beyond translation, with roles in human disease, cancer metabolism, and mitochondrial function [1,2,3,7]. Understanding its mechanism and regulation offers opportunities for therapeutic intervention. EDITGENE provides the tools to dissect this pathway with precision.
References
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- 2. Ju J et al.. 2024. The alanyl-tRNA synthetase AARS1 moonlights as a lactyltransferase to promote YAP signaling in gastric cancer.. J Clin Invest 134(10) PMID: 38512451
- 3. Jennings MJ et al.. 2022. NCAM1 and GDF15 are biomarkers of Charcot-Marie-Tooth disease in patients and mice.. Brain 145(11):3999-4015 PMID: 35148379
- 4. Onoguchi M et al.. 2024. Elucidation of productive alanine recognition mechanism by Escherichia coli alanyl-tRNA synthetase.. Biosystems 237:105152 PMID: 38346553
- 5. Mukai T. 2020. Rational Design of Aptamer-Tagged tRNAs.. Int J Mol Sci 21(20) PMID: 33096801
- 6. Bonnefond L et al.. 2005. Evolution of the tRNA(Tyr)/TyrRS aminoacylation systems.. Biochimie 87(9-10):873-83 PMID: 16164994
- 7. Zhang H et al.. 2021. The uniqueness of AlaRS and its human disease connections.. RNA Biol 18(11):1501-1511 PMID: 33317386
- 8. Freist W et al.. 1996. Glycyl-tRNA synthetase.. Biol Chem Hoppe Seyler 377(6):343-56 PMID: 8839980
