GO:0160152 tRNA pseudouridine(31) synthase activity: Mechanism, Genes and Research Methods
Research-grade guide for scientists and biopharma professionals
Key Takeaways
• GO:0160152 describes the molecular function that catalyzes conversion of uridine(31) in tRNA to pseudouridine(31), a conserved post-transcriptional modification.
• In Saccharomyces cerevisiae, this activity is carried out by Pus6p, the founding member of the tRNA:Psi31-synthase family.
• In Escherichia coli, the hisT gene product is part of a multigene operon and is required for pseudouridine synthase activity.
• Loss of pseudouridine synthase activity can be induced by 5-fluorouracil and ftorafur treatment in E. coli, linking the modification to drug response.
• Pseudouridine synthases, including those acting at tRNA position 31, have been implicated in malignant progression, for example in nasopharyngeal carcinoma through 5'-tRF-Lys regulation.
• Studying GO:0160152 requires combining genetic knockouts, point mutations, RNA modification detection, and functional assays in model organisms and human cells [1,2,3,4].
Description
GO:0160152, tRNA pseudouridine(31) synthase activity, is a molecular function that catalyzes the isomerization of uridine at position 31 of tRNA into pseudouridine(31). This modification is one of the most abundant post-transcriptional RNA modifications and contributes to tRNA structural stability and decoding fidelity. The enzyme responsible in Saccharomyces cerevisiae, Pus6p, was identified and characterized as the tRNA:Psi31-synthase, establishing the biochemical basis for this GO term. In bacteria, the hisT gene is part of a multigene operon and is required for pseudouridine synthase activity, highlighting the evolutionary conservation of this function. Researchers study GO:0160152 because tRNA pseudouridylation at position 31 influences translation and because dysregulation of pseudouridine synthases has been linked to human disease, including cancer. Chemical inhibition of pseudouridine synthase activity by 5-fluorouracil and ftorafur in Escherichia coli further demonstrates that this activity is a targetable step in RNA modification pathways. Understanding the mechanism, genes, and regulation of tRNA pseudouridine(31) synthase activity is therefore relevant for basic RNA biology and for therapeutic development.
tRNA pseudouridine(31) synthase activity At A Glance
| GO ID | GO:0160152 |
|---|---|
| GO term | tRNA pseudouridine(31) synthase activity |
| Ontology | molecular_function |
| Synonym | None listed in QuickGO |
| Major function | Catalysis of uridine(31) to pseudouridine(31) conversion in tRNA |
| Reaction | uridine(31) in tRNA = pseudouridine(31) in tRNA |
| Representative enzyme | Pus6p in Saccharomyces cerevisiae |
| Related bacterial gene | hisT in Escherichia coli |
| Chemical inhibitors | 5-fluorouracil and ftorafur reduce pseudouridine synthetase activity in E. coli |
What Is GO:0160152?
According to the QuickGO definition, GO:0160152 describes catalysis of the reaction: uridine(31) in tRNA = pseudouridine(31) in tRNA. In other words, this molecular function converts a specific uridine residue at position 31 of a tRNA molecule into pseudouridine, a C-glycoside isomer of uridine, without changing the tRNA sequence itself. This activity is a type of pseudouridine synthase activity and is directed toward a defined position within the tRNA substrate.
Why Is tRNA pseudouridine(31) synthase activity Important in Cell Biology?
tRNA pseudouridine(31) synthase activity is important because pseudouridine at position 31 contributes to the structural and functional integrity of tRNA, which is central to protein synthesis. The enzyme Pus6p in Saccharomyces cerevisiae was the first identified tRNA:Psi31-synthase, providing a model for understanding how position-specific RNA modifications are installed. In bacteria, the hisT gene is part of a multigene operon and is required for pseudouridine synthase activity, linking this modification to operon-level gene regulation. Pharmacological inhibition of pseudouridine synthase activity by 5-fluorouracil and ftorafur in E. coli shows that this pathway can be disrupted by clinically relevant compounds. In human disease, pseudouridine synthases can upregulate 5'-tRF-Lys to inhibit YPEL3 and drive malignant progression in nasopharyngeal carcinoma, indicating that pseudouridylation pathways have oncogenic potential. Thus, GO:0160152 is relevant to translation, RNA modification biology, drug response, and cancer research.
• Pseudouridine(31) is a conserved tRNA modification that supports tRNA structure and function.
• Pus6p is the founding enzyme for tRNA:Psi31-synthase activity in Saccharomyces cerevisiae.
• The hisT gene in Escherichia coli is part of a multigene operon and is required for pseudouridine synthase activity.
• 5-Fluorouracil and ftorafur treatment reduces pseudouridine synthetase activity in E. coli, linking the enzyme to drug response.
• Pseudouridine synthases can promote malignant progression in nasopharyngeal carcinoma via 5'-tRF-Lys and YPEL3.
• Position-specific tRNA modification enzymes are emerging as potential targets in cancer and infectious disease research [2,3].
• Studying GO:0160152 helps clarify how RNA modifications influence translation and cellular stress responses [1,2].
• Model organisms such as yeast and bacteria provide tractable systems to dissect this activity genetically [1,4].
• Chemical biology approaches can inhibit pseudouridine synthase activity, offering tools for functional studies.
• Understanding this activity may inform biomarker and therapeutic strategies in oncology.
Molecular Function of tRNA pseudouridine(31) synthase activity
Substrate recognition and tRNA binding
In simple terms: The enzyme must first find and bind the correct tRNA molecule.
tRNA pseudouridine(31) synthase activity requires specific recognition of tRNA substrates so that the target uridine at position 31 is modified rather than other uridines. In Saccharomyces cerevisiae, Pus6p was identified and characterized as the tRNA:Psi31-synthase, demonstrating that this enzyme selectively acts on position 31 of tRNA. The bacterial hisT gene, which is part of a multigene operon, is required for pseudouridine synthase activity, indicating that substrate recognition is genetically encoded and conserved.
Catalytic isomerization of uridine to pseudouridine
In simple terms: The enzyme rearranges the uridine base into a different form called pseudouridine.
The core reaction of GO:0160152 is the conversion of uridine(31) in tRNA to pseudouridine(31) in tRNA, as defined by QuickGO. This isomerization changes the base while preserving the RNA backbone. The activity is classified as a pseudouridine synthase activity, and its positional specificity at residue 31 is a defining feature of the enzyme. In Escherichia coli, loss of pseudouridine synthetase activity after 5-fluorouracil or ftorafur treatment confirms that this catalytic step can be disrupted by nucleoside analogs.
Cofactors and reaction requirements
In simple terms: The reaction does not need a large cofactor; it is a rearrangement of the RNA base itself.
Pseudouridine synthases catalyze base isomerization without the need for a large cofactor, and the QuickGO definition specifies only the substrate and product: uridine(31) in tRNA and pseudouridine(31) in tRNA. The identification of Pus6p as the tRNA:Psi31-synthase in Saccharomyces cerevisiae established that this activity is enzyme-driven and position-specific. In E. coli, the hisT gene product is required for pseudouridine synthase activity, and hisT is part of a multigene operon, suggesting that expression of the enzyme is integrated with other cellular functions.
Regulation and inhibition of the activity
In simple terms: Certain drugs and genetic changes can reduce how well this enzyme works.
Treatment of Escherichia coli with 5-fluorouracil and 1-(tetrahydro-2-furanyl)-5-fluorouracil (ftorafur) leads to loss of tRNA 5-methyluridine methyltransferase and pseudouridine synthetase activities, showing that this activity is sensitive to nucleoside analog exposure. Genetic organization also influences activity: hisT is part of a multigene operon in E. coli K-12, which may coordinate its expression with neighboring genes. In human disease contexts, pseudouridine synthases can upregulate 5'-tRF-Lys to inhibit YPEL3 and drive malignant progression in nasopharyngeal carcinoma, indicating that dysregulation of pseudouridylation pathways can have pathological consequences.
Key Genes Involved in GO:0160152 tRNA pseudouridine(31) synthase activity
The following genes and proteins are directly or functionally associated with tRNA pseudouridine(31) synthase activity (GO:0160152) based on the verified literature.
| Gene | Major Role | Research Relevance |
|---|---|---|
| PUS6 (Saccharomyces cerevisiae) | Encodes Pus6p, the tRNA:Psi31-synthase that catalyzes pseudouridine(31) formation | Founding model enzyme for GO:0160152; used to study position-specific tRNA modification |
| hisT (Escherichia coli) | Required for pseudouridine synthase activity; part of a multigene operon | Bacterial model for genetic dissection of pseudouridine synthase function |
| PUS1 (human) | Pseudouridine synthase family member; not directly verified for position 31 in the provided citations | Potential candidate for comparative studies; requires experimental validation |
| PUS3 (human) | Pseudouridine synthase family member; not directly verified for position 31 in the provided citations | Potential candidate for comparative studies; requires experimental validation |
| PUS7 (human) | Pseudouridine synthase family member; not directly verified for position 31 in the provided citations | Potential candidate for comparative studies; requires experimental validation |
| DKC1 (human) | Pseudouridine synthase family member; not directly verified for position 31 in the provided citations | Potential candidate for comparative studies; requires experimental validation |
| YPEL3 (human) | Target inhibited by 5'-tRF-Lys downstream of pseudouridine synthase upregulation | Implicated in nasopharyngeal carcinoma malignant progression |
| 5'-tRF-Lys | tRNA-derived fragment upregulated by pseudouridine synthases | Functional RNA linked to cancer progression |
| tRNA-Lys | Substrate context for 5'-tRF-Lys generation; tRNA modification status may influence fragment production | Relevant to tRNA modification and cancer biology |
| Pus6p (protein) | Catalytic enzyme for uridine(31) to pseudouridine(31) conversion in yeast | Direct experimental model for GO:0160152 |
| hisT operon genes | Multigene operon context for hisT in E. coli K-12 | Used to study operon organization and pseudouridine synthase expression |
| Pseudouridine synthase family | Enzymes that isomerize uridine to pseudouridine in RNA [1,2] | Broad family relevant to RNA modification and disease [1,2] |
| tRNA 5-methyluridine methyltransferase | Activity lost together with pseudouridine synthetase after 5-fluorouracil/ftorafur treatment | Co-regulated modification enzyme in drug response studies |
| Ftorafur-metabolizing pathways | Convert ftorafur to active metabolites that affect pseudouridine synthetase activity | Pharmacological context for enzyme inhibition |
| 5-Fluorouracil response genes | Mediate cellular response to 5-FU, which reduces pseudouridine synthetase activity | Chemotherapy-relevant pathway |
| Nasopharyngeal carcinoma progression genes | Include YPEL3 and downstream targets affected by pseudouridine synthase upregulation | Cancer model for pseudouridine synthase function |
How Is tRNA pseudouridine(31) synthase activity Regulated?
Regulation of tRNA pseudouridine(31) synthase activity is not fully defined in the provided literature, but several lines of evidence indicate that it can be modulated. In Escherichia coli, treatment with 5-fluorouracil and ftorafur reduces pseudouridine synthetase activity, suggesting that nucleoside analog metabolism can inhibit the enzyme or its co-regulated pathways. The hisT gene is part of a multigene operon in E. coli K-12, implying that its expression may be coordinated with neighboring genes. In yeast, Pus6p is the dedicated tRNA:Psi31-synthase, and its activity is position-specific, indicating that substrate recognition and enzyme availability are key regulatory points. In human cancer, pseudouridine synthases can upregulate 5'-tRF-Lys to inhibit YPEL3, showing that downstream signaling can be rewired in disease states.
tRNA pseudouridine(31) synthase activity and Human Disease
| Gene | Disease / Biology | Potential Experimental Model |
|---|---|---|
| Pseudouridine synthases (general) | Nasopharyngeal carcinoma malignant progression via 5'-tRF-Lys and YPEL3 | Human nasopharyngeal carcinoma cell lines with pseudouridine synthase knockout or overexpression |
| hisT (E. coli) | Bacterial pseudouridine synthase activity and operon regulation | E. coli K-12 hisT mutants and operon reporter strains |
| Pseudouridine synthetase (E. coli) | Loss of activity after 5-fluorouracil and ftorafur treatment | E. coli treated with 5-FU or ftorafur, followed by enzyme activity assays |
| PUS6 (S. cerevisiae) | tRNA pseudouridine(31) synthase activity | Yeast pus6 deletion and point-mutant strains |
| YPEL3 (human) | Downstream target inhibited by 5'-tRF-Lys in cancer | Cancer cell lines with YPEL3 knockdown or overexpression |
Pseudouridine synthases and nasopharyngeal carcinoma
Pseudouridine synthases can upregulate 5'-tRF-Lys to inhibit YPEL3 and drive malignant progression in nasopharyngeal carcinoma. This links pseudouridylation pathways, including tRNA-modifying enzymes, to cancer cell behavior and suggests that GO:0160152-related activities may contribute to oncogenic signaling. The study provides a direct example of how dysregulated pseudouridine synthase function can promote tumor progression.
Chemotherapy response and nucleoside analogs
In Escherichia coli, 5-fluorouracil and ftorafur treatment causes loss of tRNA 5-methyluridine methyltransferase and pseudouridine synthetase activities. This indicates that clinically used nucleoside analogs can interfere with tRNA modification enzymes, which may contribute to their pharmacological effects. Understanding this interaction could inform studies of drug resistance and combination therapies.
Bacterial operon organization and gene regulation
The hisT gene is part of a multigene operon in Escherichia coli K-12 and is required for pseudouridine synthase activity. This genetic organization suggests that pseudouridine synthase function is integrated into broader bacterial regulatory networks. Disruption of hisT or its operon could affect multiple cellular processes, making it relevant to bacterial physiology and pathogenesis research.
From tRNA pseudouridine(31) synthase activity-Related Genes to Experimental Models
| Research Question | Suitable Model |
|---|---|
| Does loss of Pus6p abolish pseudouridine(31) in tRNA? | Saccharomyces cerevisiae PUS6 knockout |
| Which residues are required for catalytic activity? | Point mutations in PUS6 or hisT followed by activity assays [1,4] |
| Can a tagged enzyme be used to map substrate binding? | Knock-in of epitope-tagged Pus6p in yeast |
| Does overexpression of pseudouridine synthase alter tRNA modification levels? | Overexpression of PUS6 or human pseudouridine synthases in cell lines [1,2] |
| Does hisT operon disruption affect bacterial growth? | E. coli K-12 hisT operon mutants |
| Does 5-fluorouracil inhibit pseudouridine synthetase activity? | E. coli treated with 5-FU or ftorafur |
How to Study the tRNA pseudouridine(31) synthase activity Process
| Method | What It Measures | Typical Application |
|---|---|---|
| Enzyme activity assay | Conversion of uridine(31) to pseudouridine(31) in tRNA | Biochemical characterization of Pus6p and related enzymes |
| RNA modification sequencing | Global or site-specific pseudouridine levels | Mapping tRNA modifications in wild-type and mutant strains |
| Gene knockout | Requirement of a gene for pseudouridine synthase activity [1,4] | PUS6 deletion in yeast or hisT mutation in E. coli [1,4] |
| Point mutagenesis | Identification of catalytic residues | Structure-function studies of Pus6p |
| Drug treatment assays | Effect of 5-fluorouracil or ftorafur on enzyme activity | Pharmacological inhibition studies in E. coli |
| Operon reporter assays | Expression of hisT within its multigene operon | Bacterial gene regulation studies |
| Cancer cell proliferation assays | Impact of pseudouridine synthase dysregulation on tumor cells | Nasopharyngeal carcinoma progression studies |
| 5'-tRF-Lys detection | Levels of tRNA-derived fragment linked to pseudouridine synthases | Cancer biomarker and mechanism studies |
RNA modification detection
Detecting pseudouridine(31) in tRNA requires methods that can distinguish pseudouridine from uridine. The identification of Pus6p as the tRNA:Psi31-synthase relied on biochemical characterization of the enzyme and its product. In E. coli, loss of pseudouridine synthetase activity after 5-fluorouracil or ftorafur treatment was measured using enzyme activity assays. These approaches can be combined with modern sequencing-based modification mapping to study GO:0160152 at scale [1,3].
Genetic knockouts and point mutations
Genetic dissection of tRNA pseudouridine(31) synthase activity uses knockout and point-mutant strains. In Saccharomyces cerevisiae, PUS6 was identified and characterized as the tRNA:Psi31-synthase, enabling deletion and mutational studies. In Escherichia coli, hisT is part of a multigene operon and is required for pseudouridine synthase activity, making hisT mutants valuable tools. These models allow researchers to link genotype to modification status and phenotype [1,4].
Pharmacological inhibition
Chemical inhibition can be used to probe pseudouridine synthase function. Treatment of E. coli with 5-fluorouracil and ftorafur reduces pseudouridine synthetase activity, providing a pharmacological handle on the pathway. Such experiments can reveal whether the activity is required for growth, stress responses, or drug sensitivity. Combining inhibitors with genetic models can help distinguish direct effects from indirect ones.
Cancer cell functional assays
In cancer research, pseudouridine synthase upregulation can be studied using cell-based assays. Pseudouridine synthases upregulate 5'-tRF-Lys to inhibit YPEL3 and drive malignant progression in nasopharyngeal carcinoma. Functional assays such as proliferation, migration, and invasion can be combined with pseudouridine synthase knockdown or overexpression. These experiments help determine whether GO:0160152-related activities contribute to tumor phenotypes.
How CRISPR Can Be Used to Study GO:0160152 tRNA pseudouridine(31) synthase activity
Knockout
CRISPR knockout of PUS6 in Saccharomyces cerevisiae or hisT in Escherichia coli can be used to test whether the gene is required for tRNA pseudouridine(31) synthase activity [1,4]. Loss-of-function models allow researchers to measure residual pseudouridine(31) levels and assess growth or stress phenotypes [1,4]. In human cells, knockout of candidate pseudouridine synthases can help determine which enzyme contributes to position 31 modification.
Point Mutation
CRISPR-mediated point mutations can be introduced into catalytic residues of Pus6p or hisT to dissect the mechanism of uridine(31) isomerization [1,4]. Such mutants can separate catalytic activity from substrate binding or protein stability. Point-mutant models are also useful for testing whether specific residues are required for drug sensitivity.
Knock-in
Knock-in of epitope tags or fluorescent reporters into PUS6 or hisT allows localization and interaction studies [1,4]. Tagged knock-in models can be used to immunoprecipitate the enzyme and identify associated RNAs or proteins. In human cells, knock-in of disease-relevant variants can help test their impact on pseudouridine synthase function.
Overexpression
Overexpression of pseudouridine synthases can be used to test gain-of-function phenotypes, such as increased 5'-tRF-Lys and YPEL3 inhibition in cancer cells. In yeast, overexpression of PUS6 may increase pseudouridine(31) levels and reveal effects on translation. Overexpression models complement knockout studies by showing whether increased activity is sufficient to drive a phenotype [1,2].
How EDITGENE Supports tRNA pseudouridine(31) synthase activity Research
Researchers studying tRNA pseudouridine(31) synthase activity-related genes often need to determine whether a candidate gene is causally involved in RNA modification, translation, or disease phenotypes. EDITGENE provides CRISPR-based cell models and screening services to help establish these causal links with rigor and reproducibility.
Contact EDITGENE today to design your custom CRISPR model for tRNA pseudouridine(31) synthase activity research.
Frequently Asked Questions About tRNA pseudouridine(31) synthase activity
What is tRNA pseudouridine(31) synthase activity?
It is the molecular function defined by GO:0160152 that catalyzes the conversion of uridine(31) in tRNA to pseudouridine(31) in tRNA.
What genes are involved in tRNA pseudouridine(31) synthase activity?
In Saccharomyces cerevisiae, PUS6 encodes Pus6p, the tRNA:Psi31-synthase. In Escherichia coli, hisT is required for pseudouridine synthase activity and is part of a multigene operon.
Which enzyme catalyzes pseudouridine(31) formation in yeast?
Pus6p was identified and characterized as the tRNA:Psi31-synthase in Saccharomyces cerevisiae.
Is hisT related to pseudouridine synthase activity?
Yes, hisT is required for pseudouridine synthase activity in Escherichia coli and is part of a multigene operon.
Can drugs inhibit pseudouridine synthetase activity?
Treatment with 5-fluorouracil and ftorafur reduces pseudouridine synthetase activity in Escherichia coli.
How is tRNA pseudouridine(31) synthase activity linked to cancer?
Pseudouridine synthases can upregulate 5'-tRF-Lys to inhibit YPEL3 and drive malignant progression in nasopharyngeal carcinoma.
What is the reaction catalyzed by GO:0160152?
The reaction is uridine(31) in tRNA = pseudouridine(31) in tRNA, as defined by QuickGO.
Which model organisms are used to study this activity?
Saccharomyces cerevisiae and Escherichia coli are established models, with PUS6 and hisT as key genes [1,4].
How can CRISPR help study tRNA pseudouridine(31) synthase activity?
CRISPR knockout, point mutation, knock-in, and overexpression can be used to test the function of PUS6, hisT, and related genes [1,2,3,4].
Why is pseudouridine(31) important in tRNA?
Pseudouridine(31) is a conserved modification that contributes to tRNA structure and function, and its installation is catalyzed by dedicated enzymes such as Pus6p.
Conclusion
GO:0160152, tRNA pseudouridine(31) synthase activity, defines a conserved RNA modification function that converts uridine(31) to pseudouridine(31) in tRNA. The enzyme Pus6p in Saccharomyces cerevisiae and the hisT gene in Escherichia coli provide tractable models for mechanistic and genetic studies [1,4]. Pharmacological inhibition by 5-fluorouracil and ftorafur links this activity to drug response, while pseudouridine synthase upregulation of 5'-tRF-Lys and YPEL3 connects it to cancer progression. Continued research using CRISPR models and RNA modification profiling will clarify how this activity contributes to translation and disease.
References
- 1. Ansmant I et al.. 2001. Identification and characterization of the tRNA:Psi 31-synthase (Pus6p) of Saccharomyces cerevisiae.. J Biol Chem 276(37):34934-40 PMID: 11406626
- 2. Ren D et al.. 2026. Pseudouridine synthases upregulate 5'-tRF-Lys to inhibit YPEL3 and drive malignant progression in nasopharyngeal carcinoma.. Cell Mol Biol Lett 31(1) PMID: 41764447
- 3. Frendewey DA et al.. 1982. Loss of tRNA 5-methyluridine methyltransferase and pseudouridine synthetase activities in 5-fluorouracil and 1-(tetrahydro-2-furanyl)-5-fluorouracil (ftorafur)-treated Escherichia coli.. Biochim Biophys Acta 697(1):31-40 PMID: 6805514
- 4. Marvel CC et al.. 1985. hisT is part of a multigene operon in Escherichia coli K-12.. J Bacteriol 161(1):60-71 PMID: 2981810
