YciO is an uncharacterized member of the PF01300 (TsaC/Sua5/YciO/YrdC) family in E. coli. It is a PARALOG of TsaC (L-threonylcarbamoyladenylate synthase, EC 2.7.7.87), but extensive evidence demonstrates that YciO does NOT perform the same biological function as TsaC. While YciO shows very weak L-threonylcarbamoyladenylate synthase activity in vitro (0.14 nM/min TC-AMP production, PMID:37963869), this is more than 4 orders of magnitude weaker than TsaC (2.8 uM/min) under similar conditions (PMID:40703034). The canonical KRSN tetrad (KxR...SxN) required for TC-AMP synthesis is replaced by KxL...SxM in YciO, consistent with altered or lost catalytic activity. In vivo experiments have shown that TsaC is essential even when yciO is present or overexpressed, indicating YciO cannot substitute for TsaC function. The weak in vitro activity is likely residual ancestral/promiscuous activity, not the biological function. YciO possesses a large conserved positively charged surface (absent in TsaC) predicted to interact with RNA (PMID:40703034). Genomic context analysis shows yciO genes frequently colocalize with rnm genes encoding RNase AM, a 5-to-3 exonuclease that matures the 5-prime ends of rRNAs. The true biological function of YciO is unknown but likely relates to rRNA metabolism rather than tRNA modification. This gene is a textbook case of how paralog confusion and uncritical acceptance of in vitro promiscuous activity can lead to incorrect functional annotations.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
|
GO:0005829
cytosol
|
IBA
GO_REF:0000033 |
ACCEPT |
Summary: IBA annotation for cytosol localization based on phylogenetic inference (PANTHER). Consistent with experimental IDA evidence from two independent proteomics studies (PMID:15911532, PMID:18304323) that identified YciO in the cytosolic fraction of E. coli K-12.
Reason: Cytosolic localization is well-supported by both phylogenetic inference and direct experimental evidence from mass spectrometry-based proteomics. Two independent studies identified YciO as a cytosolic protein.
Supporting Evidence:
PMID:15911532
Here we describe a proteomic analysis of Escherichia coli in which 3,199 protein forms were detected, and of those 2,160 were annotated and assigned to the cytosol, periplasm, inner membrane, and outer membrane by biochemical fractionation followed by two-dimensional gel electrophoresis and tandem mass spectrometry.
PMID:18304323
Here, we describe an experimental scheme to maximize the coverage of proteins identified by mass spectrometry of a complex biological sample.
|
|
GO:0003725
double-stranded RNA binding
|
IEA
GO_REF:0000002 |
UNDECIDED |
Summary: IEA annotation based on InterPro domain IPR006070 (Sua5-like domain) mapping to dsRNA binding. This is a computational prediction from InterPro. While YciO does possess a large conserved positively charged surface predicted to interact with RNA (PMID:40703034), the specific annotation as dsRNA binding is not experimentally validated and the InterPro mapping may be overly specific. The positively charged surface is a feature distinguishing YciO from TsaC, suggesting RNA interaction is plausible, but the specific type of RNA (dsRNA vs rRNA) is uncertain.
Reason: The InterPro-based prediction of dsRNA binding is plausible given the large positively charged surface unique to YciO proteins (PMID:40703034), but there is no direct experimental evidence for dsRNA binding specifically. The genomic context (colocalization with rnm genes encoding RNase AM, a rRNA maturation enzyme) suggests rRNA interaction may be more likely. Without experimental evidence, it is difficult to determine whether dsRNA binding or a more specific RNA binding term is appropriate. The annotation should be revisited when experimental data on YciO RNA binding become available.
Supporting Evidence:
PMID:40703034
the structure of YciO exhibits a large positively charged surface predicted to interact with RNA
PMID:40703034
genes are colocalized with rnm genes (Fig. 7), which encode the recently characterized RNase AM, a 5′ to 3′ exonuclease that matures the 5′ end of all 3 ribosomal RNAs in E. coli
|
|
GO:0061710
L-threonylcarbamoyladenylate synthase activity
|
IEA
GO_REF:0000003 |
REMOVE |
Summary: IEA annotation based on EC number mapping (EC:2.7.7.87). This EC number was assigned to YciO in UniProt based on the Kim et al. 2023 deep learning prediction and in vitro assay (PMID:37963869). However, de Crecy-Lagard et al. 2025 (PMID:40703034) demonstrated that this is a classic case of paralog incorrect (PLI) annotation. YciO is a paralog of TsaC but does not perform the same biological function. The in vitro activity is 4 orders of magnitude weaker than TsaC and represents residual ancestral/promiscuous activity. In vivo experiments show TsaC is essential even with yciO present. The EC2GO mapping propagates this incorrect EC assignment.
Reason: This annotation propagates through EC2GO from the incorrect EC:2.7.7.87 assignment to YciO. De Crecy-Lagard et al. 2025 (PMID:40703034) provide compelling evidence that YciO does NOT function as an L-threonylcarbamoyladenylate synthase in vivo. The weak in vitro activity (0.14 nM/min vs 2.8 uM/min for TsaC) is residual ancestral activity, not the biological function. In vivo, TsaC is essential even when yciO is overexpressed. The EC number 2.7.7.87 prediction was given a confidence score of 0 (refuted) by expert curation in the de Crecy-Lagard study.
Supporting Evidence:
PMID:40703034
YciO does not perform the same function as TsaC/Susa5 in vivo experiments
PMID:40703034
the activity reported (0.14 nM/min TC-AMP production rate) for E. coli YciO is more than 4 orders of magnitude weaker than that of E. coli TsaC (2.8 μM/min) at the same enzyme concentration and similar reaction conditions
PMID:40703034
the functional puzzle is far from being solved for proteins of the YciO subgroup, and even if the existing data suggest a role in RNA metabolism, it cannot be the same as TsaC, and the EC number 2.7.7.87 prediction was given a CS of 0
|
|
GO:0061710
L-threonylcarbamoyladenylate synthase activity
|
IDA
PMID:37963869 Functional annotation of enzyme-encoding genes using deep le... |
REMOVE |
Summary: IDA annotation based on Kim et al. 2023 (PMID:37963869), which used DeepECTransformer to predict EC 2.7.7.87 for YciO and validated it with an in vitro enzyme assay showing a specific activity of 0.0705 U/mg. However, de Crecy-Lagard et al. 2025 (PMID:40703034) demonstrated this is a paralog incorrect (PLI) prediction. The measured activity (0.14 nM/min TC-AMP) is more than 4 orders of magnitude weaker than genuine TsaC activity (2.8 uM/min). This weak activity represents residual ancestral/promiscuous catalytic activity from shared evolutionary origin, not the biological function. In vivo experiments show YciO cannot substitute for TsaC. Models of enzyme evolution predict that paralogs retain promiscuous ancestral activities detectable in vitro. The IDA evidence code is technically correct for in vitro detection, but the annotation misrepresents the biological function of YciO.
Reason: While Kim et al. 2023 (PMID:37963869) did detect L-threonylcarbamoyladenylate synthase activity in vitro, de Crecy-Lagard et al. 2025 (PMID:40703034) conclusively demonstrate this is promiscuous/residual ancestral activity, not the biological function. The activity is 4 orders of magnitude weaker than TsaC. In vivo, TsaC is essential even with YciO present or overexpressed. The Kim et al. study used the in vitro assay to validate a deep learning prediction without considering the in vivo context, genomic evidence, or the distinction between promiscuous and biological activity. As stated in PMID:40703034: in vitro activity alone is not sufficient to validate protein function in vivo. This annotation should be removed as it represents an over-annotation based on promiscuous activity of a paralog.
Supporting Evidence:
PMID:37963869
In the case of YciO, which was previously annotated to belong to the SUA5 family, DeepECtransformer predicted its EC number to be EC:2.7.7.87 (L-threonylcarbamoyladenylate synthase) with the prediction score of 0.9108. The specific activity of YciO was measured to be 0.0705 U mg-1
PMID:40703034
the activity reported (0.14 nM/min TC-AMP production rate) for E. coli YciO is more than 4 orders of magnitude weaker than that of E. coli TsaC (2.8 μM/min) at the same enzyme concentration and similar reaction conditions ... consistent with the possibility of a missing partner or a different biological substrate for YciO
PMID:40703034
YciO does not perform the same function as TsaC/Susa5 in vivo experiments
PMID:40703034
TsaC is an enzyme predicted to have been present in the Last Universal Common Ancestor ... YciO is a likely paralog of TsaC (Fig. 6), and as such, it is likely to have residual ancestral catalytic activity that can be detected in vitro
PMID:40703034
in vitro activity alone is not sufficient to validate the function of a protein in vivo
file:ECOLI/yciO/yciO-deep-research-falcon.md
Falcon deep research independently confirms YciO is not part of the canonical t6A pathway. Multiple sources (Harris 2015, Thiaville 2014, Carvalho 2017, Pichard-Kostuch 2023) establish YciO as a divergent paralog lacking the conserved KRSN tetrad required for TC-AMP synthesis.
|
|
GO:0005829
cytosol
|
IDA
PMID:15911532 Localization, annotation, and comparison of the Escherichia ... |
ACCEPT |
Summary: IDA annotation for cytosol localization from Lopez-Campistrous et al. 2005 (PMID:15911532), a large-scale proteomics study of E. coli K-12 that assigned proteins to subcellular compartments by biochemical fractionation followed by 2D gel electrophoresis and tandem mass spectrometry.
Reason: Direct experimental evidence from proteomics-based subcellular fractionation. The study identified 2,160 protein forms and assigned them to cytosol, periplasm, inner membrane, and outer membrane. YciO is a soluble cytoplasmic protein consistent with this localization.
Supporting Evidence:
PMID:15911532
Here we describe a proteomic analysis of Escherichia coli in which 3,199 protein forms were detected, and of those 2,160 were annotated and assigned to the cytosol, periplasm, inner membrane, and outer membrane by biochemical fractionation followed by two-dimensional gel electrophoresis and tandem mass spectrometry.
|
|
GO:0005829
cytosol
|
IDA
PMID:18304323 Protein abundance profiling of the Escherichia coli cytosol. |
ACCEPT |
Summary: IDA annotation for cytosol localization from Ishihama et al. 2008 (PMID:18304323), a comprehensive protein abundance profiling study of the E. coli cytosol using mass spectrometry.
Reason: Independent experimental confirmation of cytosolic localization from a second large-scale proteomics study. Consistent with the IDA annotation from PMID:15911532 and the IBA annotation.
Supporting Evidence:
PMID:18304323
Here, we describe an experimental scheme to maximize the coverage of proteins identified by mass spectrometry of a complex biological sample.
|
|
GO:0003723
RNA binding
|
ISS
PMID:40703034 Limitations of current machine learning models in predicting... |
NEW |
Summary: NEW annotation. YciO possesses a large conserved positively charged surface (comprising ~6% of total molecular surface area) that is present and conserved in all YciO family members but absent in TsaC proteins (PMID:40703034). This surface is predicted to interact with RNA. Genomic context shows yciO genes colocalize with rnm genes (encoding RNase AM, a rRNA maturation enzyme), suggesting involvement in rRNA metabolism.
Reason: De Crecy-Lagard et al. 2025 (PMID:40703034) provide structural and genomic context evidence that YciO has a conserved positively charged surface predicted to bind RNA. While the specific RNA target has not been experimentally determined, general RNA binding is well supported by structural analysis. This is proposed as a more appropriate molecular function annotation than the incorrect L-threonylcarbamoyladenylate synthase activity.
Supporting Evidence:
PMID:40703034
the structure of YciO exhibits a large positively charged surface predicted to interact with RNA
PMID:40703034
This large positively charged surface is conserved in YciO proteins and is absent in TsaC proteins
|
Q: What is the actual RNA substrate of YciO?
Q: Does YciO physically interact with RNase AM (Rnm) or other rRNA processing factors?
Q: Should the UniProt record for YciO (P0AFR4) be corrected to remove the EC 2.7.7.87 assignment?
Experiment: RNA co-immunoprecipitation or CLIP-seq to identify RNA binding partners of YciO. YciO has a conserved positively charged surface predicted to bind RNA. Identifying the actual RNA targets (rRNA, tRNA, mRNA, or other) would resolve the functional puzzle.
Hypothesis: YciO binds rRNA based on its conserved positively charged surface and genomic colocalization with rnm (RNase AM) genes.
Experiment: In vivo complementation tests with yciO deletion in combination with rnm deletion. Genomic context suggests functional coupling between YciO and RNase AM. A synthetic interaction would support the hypothesis that YciO participates in rRNA maturation.
Hypothesis: YciO and RNase AM (Rnm) function in the same rRNA maturation pathway, and double deletion will show a synthetic phenotype.
Experiment: Electrophoretic mobility shift assays (EMSAs) with various RNA substrates including rRNA fragments, tRNA, and dsRNA to narrow down the RNA binding specificity suggested by the positively charged surface.
Hypothesis: YciO preferentially binds rRNA over other RNA species.
YciO DeepECTF prediction review. The DeepECTF prediction of L-threonylcarbamoyladenylate synthase (EC 2.7.7.87) is incorrect - a classic paralog overannotation error. YciO is a paralog of TsaC but shows 10,000x weaker activity and does not complement tsaC mutants in vivo. Structural and genomic context evidence suggests a distinct role in rRNA metabolism.
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model: Edison Scientific Literature
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start_time: '2026-03-22T17:47:50.390696'
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template_variables:
organism: ECOLI
gene_id: yciO
gene_symbol: yciO
uniprot_accession: P0AFR4
protein_description: 'RecName: Full=Threonylcarbamoyl-AMP synthase YciO {ECO:0000305|PubMed:37963869};
EC=2.7.7.87 {ECO:0000269|PubMed:37963869}; AltName: Full=L-threonylcarbamoyladenylate
synthase {ECO:0000303|PubMed:37963869};'
gene_info: Name=yciO; OrderedLocusNames=b1267, JW5196;
organism_full: Escherichia coli (strain K12).
protein_family: Belongs to the SUA5 family. .
protein_domains: DHBP_synth_RibB-like_a/b_dom. (IPR017945); Sua5-like_dom. (IPR006070);
SUA5_domain. (IPR052532); Sua5_yciO_yrdC (PF01300)
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BEFORE YOU BEGIN RESEARCH: You MUST verify you are researching the CORRECT gene/protein. Gene symbols can be ambiguous, especially for less well-characterized genes from non-model organisms.
DO NOT PROCEED WITH RESEARCH ON A DIFFERENT GENE. Instead:
- State clearly: "The gene symbol 'yciO' is ambiguous or literature is limited for this specific protein"
- Explain what you found (e.g., "Found extensive literature on a different gene with the same symbol in a different organism")
- Describe the protein based ONLY on the UniProt information provided above
- Suggest that the protein function can be inferred from domain/family information
Please provide a comprehensive research report on the gene yciO (gene ID: yciO, UniProt: P0AFR4) in ECOLI.
The research report should be a detailed narrative explaining the function, biological processes, and localization of the gene product. Citations should be given for all claims.
You should prioritize authoritative reviews and primary scientific literature when conducting research. You can supplement
this with annotations you find in gene/protein databases, but these can be outdated or inaccurate.
We are specifically interested in the primary function of the gene - for enzymes, what reaction is catalyzed, and what is the substrate specificity? For transporters, what is the substrate? For structural proteins or adapters, what is the broader structural role? For signaling molecules, what is the role in the pathway.
We are interested in where in or outside the cell the gene product carries out its function.
We are also interested in the signaling or biochemical pathways in which the gene functions. We are less interested in broad pleiotropic effects, except where these elucidate the precise role.
Include evidence where possible. We are interested in both experimental evidence as well as inference from structure, evolution, or bioinformatic analysis. Precise studies should be prioritized over high-throughput, where available.
Question: You are an expert researcher providing comprehensive, well-cited information.
Provide detailed information focusing on:
1. Key concepts and definitions with current understanding
2. Recent developments and latest research (prioritize 2023-2024 sources)
3. Current applications and real-world implementations
4. Expert opinions and analysis from authoritative sources
5. Relevant statistics and data from recent studies
Format as a comprehensive research report with proper citations. Include URLs and publication dates where available.
Always prioritize recent, authoritative sources and provide specific citations for all major claims.
BEFORE YOU BEGIN RESEARCH: You MUST verify you are researching the CORRECT gene/protein. Gene symbols can be ambiguous, especially for less well-characterized genes from non-model organisms.
DO NOT PROCEED WITH RESEARCH ON A DIFFERENT GENE. Instead:
- State clearly: "The gene symbol 'yciO' is ambiguous or literature is limited for this specific protein"
- Explain what you found (e.g., "Found extensive literature on a different gene with the same symbol in a different organism")
- Describe the protein based ONLY on the UniProt information provided above
- Suggest that the protein function can be inferred from domain/family information
Please provide a comprehensive research report on the gene yciO (gene ID: yciO, UniProt: P0AFR4) in ECOLI.
The research report should be a detailed narrative explaining the function, biological processes, and localization of the gene product. Citations should be given for all claims.
You should prioritize authoritative reviews and primary scientific literature when conducting research. You can supplement
this with annotations you find in gene/protein databases, but these can be outdated or inaccurate.
We are specifically interested in the primary function of the gene - for enzymes, what reaction is catalyzed, and what is the substrate specificity? For transporters, what is the substrate? For structural proteins or adapters, what is the broader structural role? For signaling molecules, what is the role in the pathway.
We are interested in where in or outside the cell the gene product carries out its function.
We are also interested in the signaling or biochemical pathways in which the gene functions. We are less interested in broad pleiotropic effects, except where these elucidate the precise role.
Include evidence where possible. We are interested in both experimental evidence as well as inference from structure, evolution, or bioinformatic analysis. Precise studies should be prioritized over high-throughput, where available.
The UniProt target provided (P0AFR4; gene yciO, locus b1267) belongs to the SUA5/TsaC (YrdC) protein family but, based on multiple independent sources, E. coli YciO is not the canonical threonylcarbamoyl-AMP synthase used in tRNA t6A biosynthesis. Instead, the active threonylcarbamoyl-AMP synthase in E. coli is TsaC, also known as YrdC, which is essential for t6A formation and viability; YciO is described as a divergent, nonessential paralog/homolog not associated with the t6A pathway, and it lacks the canonical active-site signature (KRSN / KxR…SxN) found in functional TsaC/Sua5 proteins. (harris2015nmrbasedstructuralanalysis pages 1-2, carvalho2017slr0006likeproteinsa pages 1-2, thiaville2014diversityofthe pages 7-7)
Accordingly, the best-supported functional annotation for E. coli yciO (P0AFR4) is: SUA5/TsaC-family protein, likely catalytically inactive or noncanonical relative to t6A TC-AMP synthesis; its precise physiological role in E. coli remains unresolved in the retrieved literature, with only tentative links (e.g., glycogen metabolism) reported. (carvalho2017slr0006likeproteinsa pages 1-2, thiaville2014diversityofthe pages 7-7)
t6A (N6-threonylcarbamoyladenosine) is a universally conserved tRNA modification at position A37 (adjacent to the anticodon) on most ANN-decoding tRNAs. It contributes to translation accuracy and stabilizes the anticodon loop (e.g., through hydrogen-bonding and stacking effects). (zheng2024molecularbasisof pages 1-2, pichardkostuch2023theuniversalsua5tsac pages 1-3)
t6A biosynthesis is generally described as a two-step pathway:
1. Formation of the activated intermediate L-threonylcarbamoyl-adenylate (TC-AMP) from L-threonine + ATP + CO2/HCO3− by TsaC/Sua5-family enzymes. (zheng2024molecularbasisof pages 1-2, pichardkostuch2023theuniversalsua5tsac pages 1-3)
2. Transfer of the threonylcarbamoyl (TC) moiety from TC-AMP onto tRNA A37 by a universal catalytic family (TsaD/Kae1-like), using bacterial TsaD–TsaB–TsaE machinery in bacteria. (pichardkostuch2023theuniversalsua5tsac pages 3-4, zheng2024molecularbasisof pages 1-2)
A key sequence feature is the KRSN tetrad / KxR…SxN motif that is conserved in functional TsaC/Sua5 proteins, whereas in E. coli YciO this signature is reported as replaced by KxL…SxM, consistent with altered binding/catalysis. (carvalho2017slr0006likeproteinsa pages 1-2, pichardkostuch2023theuniversalsua5tsac media 03316335, thiaville2014diversityofthe pages 7-7)
Multiple sources explicitly support that E. coli YciO is not part of the canonical t6A37 pathway, despite structural similarity to TsaC family proteins:
- A structural study on E. coli TsaC (YrdC) notes that E. coli YciO has a solved structure but states that YciO is not associated with the t6A37 pathway. (harris2015nmrbasedstructuralanalysis pages 1-2)
- Comparative genomics and motif analysis describe YciO as a paralog lacking the conserved KRSN tetrad and “does not have the same function.” (thiaville2014diversityofthe pages 7-7)
- A phylogenetic/motif analysis emphasizes YciO’s divergence and nonessentiality, and inability to compensate for TsaC loss, consistent with functional separation. (carvalho2017slr0006likeproteinsa pages 1-2)
Conclusion: based on retrieved evidence, the primary biochemical role (EC 2.7.7.87) belongs to E. coli TsaC/YrdC, not to the UniProt target YciO (P0AFR4). (harris2015nmrbasedstructuralanalysis pages 1-2, lauhon2012mechanismofn6threonylcarbamoyladenonsine pages 1-2, zheng2024molecularbasisof pages 1-2)
The function of YciO in E. coli is described as unknown in the family-focused literature, with one report noting YciO has been linked to glycogen metabolism, but without a definitive biochemical assignment in the provided excerpts. (carvalho2017slr0006likeproteinsa pages 1-2)
No retrieved text evidence directly establishes subcellular localization of E. coli YciO. However, the relevant chemistry (tRNA maturation and t6A installation) is cytosolic in bacteria, and eukaryotic studies emphasize cytosolic localization as the primary locus for Sua5/KEOPS-dependent t6A function. (choi2024sua5catalyzinguniversal pages 1-2)
A 2023 synthesis combining phylogeny plus sequence/structure comparison concludes that:
- Sua5/TsaC proteins are ubiquitous and perform an essential function (t6A biogenesis) across life. (Published June 2023; https://doi.org/10.3389/fmicb.2023.1204045) (pichardkostuch2023theuniversalsua5tsac pages 1-3)
- TsaC likely arose multiple times from Sua5 via loss of the additional SUA5 domain, with adaptive mutations affecting substrate binding. (pichardkostuch2023theuniversalsua5tsac pages 1-3)
- The authors also discuss the rarity of co-occurrence and mention YciO as an inactive TsaC paralog in bacterial lineages. (pichardkostuch2023theuniversalsua5tsac pages 10-12)
This paper provides modern “expert synthesis” context for interpreting yciO/P0AFR4 as a divergent member within a universally conserved enzyme family, helping prevent misannotation. (pichardkostuch2023theuniversalsua5tsac pages 10-12, pichardkostuch2023theuniversalsua5tsac pages 1-3)
A 2024 Nucleic Acids Research article reiterates the conserved chemistry and bacterial pathway organization:
- TsaC/Sua5 catalyze TC-AMP formation from L-threonine, CO2/HCO3−, and ATP, and TsaDBE transfers the TC moiety to tRNA A37 in bacteria. (Published March 2024; https://doi.org/10.1093/nar/gkae179) (zheng2024molecularbasisof pages 1-2)
While not E. coli yciO-specific, this provides current authoritative pathway definitions and supports accurate functional placement of the active enzyme (YrdC/TsaC) relative to the YciO paralog. (zheng2024molecularbasisof pages 1-2)
A 2024 mSphere study in the fungal pathogen Cryptococcus neoformans used reverse genetics to show that loss of Sua5 (TC-AMP-forming step) recapitulates many KEOPS-related phenotypes, linking t6A biogenesis to growth, stress response, sexual development, and virulence-associated traits; notably, a Sua5 allele lacking a mitochondrial targeting sequence restored phenotypes, suggesting the primary functional locus of Sua5 is cytosolic. (Published Jan 2024; https://doi.org/10.1128/msphere.00557-23) (choi2024sua5catalyzinguniversal pages 1-2)
Although eukaryotic, this strengthens the general expert view that perturbing the TC-AMP/t6A axis has broad phenotypic consequences, motivating antimicrobial/antifungal interest in t6A machinery. (choi2024sua5catalyzinguniversal pages 1-2)
A 2024 study on TsaB/YeaZ (a bacterial t6A pathway component interacting with TsaD/Gcp) explicitly frames it as a promising antibacterial target because it is indispensable for bacterial survival, highly conserved across bacteria, and lacks eukaryotic orthologs—factors that support selectivity. (Published Apr 25, 2024; https://doi.org/10.3390/antibiotics13050393) (guo2024newinsightsinto pages 1-2)
This work also connects depletion of TsaB/YeaZ to altered morphology and global transcriptional effects, reinforcing the potential system-level consequences of inhibiting t6A-related proteins. (guo2024newinsightsinto pages 9-11, guo2024newinsightsinto pages 11-12)
Note: The retrieved 2024 antibacterial-target evidence is strongest for TsaB/YeaZ (and interacting TsaD/Gcp), not specifically for E. coli YciO, which remains of unknown function and nonessential. (carvalho2017slr0006likeproteinsa pages 1-2, guo2024newinsightsinto pages 1-2)
A reannotation across 9,176 bacterial genomes reported the following frequencies for TC-AMP synthase family members:
- TsaC present in 6,745 genomes (73%)
- TsaC2 present in 2,846 genomes (31%)
- Both TsaC and TsaC2 present in 859 genomes (9%)
- YciO present in ~54% of genomes analyzed. (Published Dec 2014; https://doi.org/10.4161/15476286.2014.992277) (thiaville2014diversityofthe pages 7-7)
These statistics support that YciO is widespread but not equivalent to TsaC/TsaC2 in function. (thiaville2014diversityofthe pages 7-7)
Biochemical characterization of TC-AMP reported that stability is highly condition-dependent:
- Under one “physiological-like” condition (pH 7.5, 37 °C, 2 mM MgCl2), TC-AMP half-life was reported as ~3.5 min. (Published Oct 2012; https://doi.org/10.1021/bi301233d) (lauhon2012mechanismofn6threonylcarbamoyladenonsine pages 1-2, lauhon2012mechanismofn6threonylcarbamoyladenonsine pages 11-12)
- Half-life examples from buffer series include 16.5 min (20 mM KPi, pH 7.5, 37 °C), and in MOPS at pH 7.5, 37 °C, half-life shortened dramatically with Mg2+ (e.g., 3.5 min at 2 mM MgCl2 down to 21 s at 20 mM MgCl2). (lauhon2012mechanismofn6threonylcarbamoyladenonsine pages 8-9)
These values support the expert interpretation that TC-AMP is a reactive intermediate whose handling may require coordinated pathway architecture (e.g., protein interactions/channeling). (lauhon2012mechanismofn6threonylcarbamoyladenonsine pages 8-9, thiaville2014diversityofthe pages 7-7)
For the active E. coli TC-AMP synthase ortholog YrdC (TsaC):
- With ATP as variable substrate (10 mM L-Thr; 20 mM bicarbonate), Km(ATP) = 93 ± 15 μM and kcat = 0.10 ± 0.02 s−1 were reported. (lauhon2012mechanismofn6threonylcarbamoyladenonsine pages 9-11)
- For the reverse reaction (TC-AMP + PPi → ATP), with TC-AMP as variable substrate (1 mM PPi), kcat = 20 ± 2 s−1, Km = 0.68 ± 0.15 μM, kcat/Km = 3.3 ± 1.1 × 10^7 M−1 s−1 were reported for YrdC. (lauhon2012mechanismofn6threonylcarbamoyladenonsine pages 9-11)
These quantitative data are for YrdC/TsaC, not for YciO (P0AFR4). (lauhon2012mechanismofn6threonylcarbamoyladenonsine pages 9-11)
| Topic | Summary | Evidence type | Year | DOI / URL | Citation |
|---|---|---|---|---|---|
| Identity of yciO in E. coli K-12 | yciO (UniProt P0AFR4; b1267) is best supported as a TsaC/Sua5 family homolog/paralog, not the canonical active TC-AMP synthase gene of E. coli. Reviews of COG0009 reannotation describe YciO as a paralog found in many bacteria. | Phylogenomics, comparative annotation | 2014 | https://doi.org/10.4161/15476286.2014.992277 | (thiaville2014diversityofthe pages 7-7) |
| Active enzyme in E. coli t6A biosynthesis | The active threonylcarbamoyl-AMP synthase in E. coli is TsaC = YrdC, not YciO. E. coli yrdC is essential for t6A formation, and YrdC/TsaC catalyzes TC-AMP formation. | Biochemistry, genetics, nomenclature review | 2012, 2014, 2015 | https://doi.org/10.1021/bi301233d; https://doi.org/10.4161/15476286.2014.992277; https://doi.org/10.1074/jbc.m114.631242 | (lauhon2012mechanismofn6threonylcarbamoyladenonsine pages 1-2, thiaville2014diversityofthe pages 2-3, harris2015nmrbasedstructuralanalysis pages 1-2) |
| Is YciO functionally associated with the t6A pathway? | Available evidence indicates YciO is not functionally associated with the canonical t6A pathway in E. coli: it is described as an inactive or functionally divergent paralog, “not associated with the t6A37 pathway,” nonessential for survival, and unable to compensate for loss of TsaC/TsaC2. | Structural comparison, family analysis, phylogenomics | 2015, 2017, 2023 | https://doi.org/10.1074/jbc.m114.631242; https://doi.org/10.1016/j.ympev.2016.12.033; https://doi.org/10.3389/fmicb.2023.1204045 | (harris2015nmrbasedstructuralanalysis pages 1-2, carvalho2017slr0006likeproteinsa pages 1-2, pichardkostuch2023theuniversalsua5tsac pages 10-12, pichardkostuch2023theuniversalsua5tsac media ef9a6773) |
| Catalytic motif in active TsaC/Sua5 proteins | Canonical TsaC/Sua5 proteins carry the KRSN tetrad / KxR…SxN motif required for TC-AMP synthesis. Figure/text summaries identify motifs such as (K/R)xR and SxN as conserved active-site determinants in TsaC/Sua5. | Comparative sequence/structure analysis | 2014, 2023 | https://doi.org/10.4161/15476286.2014.992277; https://doi.org/10.3389/fmicb.2023.1204045 | (thiaville2014diversityofthe pages 7-7, pichardkostuch2023theuniversalsua5tsac media 03316335) |
| Motif divergence in YciO | In YciO, the canonical active-site motif is diverged: the TsaC/TsaC2 KxR…SxN signature is reported as replaced by KxL…SxM in E. coli YciO, consistent with loss or alteration of canonical TC-AMP synthase activity. | Sequence motif comparison | 2017 | https://doi.org/10.1016/j.ympev.2016.12.033 | (carvalho2017slr0006likeproteinsa pages 1-2) |
| Canonical bacterial t6A pathway: step 1 | Step 1: TsaC/YrdC synthesizes L-threonylcarbamoyladenylate (TC-AMP) from L-threonine + ATP + CO2/HCO3−. | Biochemistry, review | 2012, 2024 | https://doi.org/10.1021/bi301233d; https://doi.org/10.1093/nar/gkae179 | (lauhon2012mechanismofn6threonylcarbamoyladenonsine pages 1-2, zheng2024molecularbasisof pages 1-2) |
| Canonical bacterial t6A pathway: step 2 | Step 2: the TsaD–TsaB–TsaE (TsaDBE) complex transfers the threonylcarbamoyl moiety from TC-AMP to A37 of ANN-decoding tRNAs to form t6A; TsaD/Kae1 is the universal catalytic family for this transfer step. | Pathway review, mechanistic synthesis | 2023, 2024 | https://doi.org/10.3389/fmicb.2023.1204045; https://doi.org/10.1093/nar/gkae179 | (pichardkostuch2023theuniversalsua5tsac pages 3-4, zheng2024molecularbasisof pages 1-2) |
| Structural/mechanistic insight for active E. coli TsaC/YrdC | NMR analysis of E. coli TsaC/YrdC identified a central substrate-binding cavity, cooperative binding of ATP and L-threonine, and conserved residues involved in adenine and threonine recognition, supporting the mechanism of TC-AMP formation. | NMR structural biochemistry | 2015 | https://doi.org/10.1074/jbc.m114.631242 | (harris2015nmrbasedstructuralanalysis pages 1-2, harris2015nmrbasedstructuralanalysis pages 8-10) |
| Evolutionary interpretation | Recent phylogenomic analysis supports Sua5 as the ancestral form and TsaC as a derived version that repeatedly arose by loss of the SUA5 domain; within this broader family, YciO is discussed as an inactive TsaC paralog in bacterial lineages. | Phylogenomics, comparative structural evolution | 2023 | https://doi.org/10.3389/fmicb.2023.1204045 | (pichardkostuch2023theuniversalsua5tsac pages 1-3, pichardkostuch2023theuniversalsua5tsac pages 10-12) |
| Practical annotation takeaway | For functional annotation of P0AFR4 / yciO, the safest evidence-based assignment is TsaC/Sua5-family homolog/paralog with likely noncanonical or inactive function relative to t6A synthesis, while the canonical E. coli t6A TC-AMP synthase is YrdC/TsaC. | Integrated interpretation from biochemistry + phylogenomics | 2012–2023 | https://doi.org/10.1021/bi301233d; https://doi.org/10.4161/15476286.2014.992277; https://doi.org/10.1074/jbc.m114.631242; https://doi.org/10.3389/fmicb.2023.1204045 | (lauhon2012mechanismofn6threonylcarbamoyladenonsine pages 1-2, thiaville2014diversityofthe pages 7-7, harris2015nmrbasedstructuralanalysis pages 1-2, pichardkostuch2023theuniversalsua5tsac pages 10-12) |
Table: This table summarizes the evidence-based distinction between E. coli K-12 yciO (P0AFR4) and the canonical active TC-AMP synthase TsaC/YrdC, along with the bacterial t6A pathway and key motif evidence. It is useful for avoiding misannotation of yciO as the primary t6A-pathway enzyme.
Recommended primary annotation (conservative, evidence-based):
- Protein family: SUA5/TsaC (YrdC) family homolog/paralog. (thiaville2014diversityofthe pages 7-7, carvalho2017slr0006likeproteinsa pages 1-2)
- Likely role: Not the canonical TC-AMP synthase in E. coli t6A biosynthesis; likely a divergent paralog lacking key catalytic motif elements. (harris2015nmrbasedstructuralanalysis pages 1-2, carvalho2017slr0006likeproteinsa pages 1-2, thiaville2014diversityofthe pages 7-7)
- Function status: Unknown / noncanonical in E. coli; reported associations include glycogen metabolism but without a definitive mechanism in the retrieved sources. (carvalho2017slr0006likeproteinsa pages 1-2)
- Localization: Not established for YciO in retrieved texts. (carvalho2017slr0006likeproteinsa pages 1-2)
References
(harris2015nmrbasedstructuralanalysis pages 1-2): Kimberly A. Harris, Benjamin G. Bobay, Kathryn L. Sarachan, Alexis F. Sims, Yann Bilbille, Christopher Deutsch, Dirk Iwata-Reuyl, and Paul F. Agris. Nmr-based structural analysis of threonylcarbamoyl-amp synthase and its substrate interactions. Journal of Biological Chemistry, 290:20032-20043, Aug 2015. URL: https://doi.org/10.1074/jbc.m114.631242, doi:10.1074/jbc.m114.631242. This article has 16 citations and is from a domain leading peer-reviewed journal.
(carvalho2017slr0006likeproteinsa pages 1-2): Leonor L. Carvalho, Tiina A. Salminen, and Käthe M. Dahlström. Slr0006-like proteins: a tsac/tsac2/ycio subfamily exclusive to cyanobacteria. Molecular phylogenetics and evolution, 109:1-10, Apr 2017. URL: https://doi.org/10.1016/j.ympev.2016.12.033, doi:10.1016/j.ympev.2016.12.033. This article has 2 citations and is from a domain leading peer-reviewed journal.
(thiaville2014diversityofthe pages 7-7): Patrick C Thiaville, Dirk Iwata-Reuyl, and Valérie de Crécy-Lagard. Diversity of the biosynthesis pathway for threonylcarbamoyladenosine (t6a), a universal modification of trna. RNA Biology, 11:1529-1539, Dec 2014. URL: https://doi.org/10.4161/15476286.2014.992277, doi:10.4161/15476286.2014.992277. This article has 114 citations and is from a peer-reviewed journal.
(zheng2024molecularbasisof pages 1-2): Xinxing Zheng, Chenchen Su, Lei Duan, Mengqi Jin, Yongtao Sun, Li Zhu, and Wenhua Zhang. Molecular basis of a. thaliana keops complex in biosynthesizing trna t6a. Nucleic Acids Research, 52:4523-4540, Mar 2024. URL: https://doi.org/10.1093/nar/gkae179, doi:10.1093/nar/gkae179. This article has 6 citations and is from a highest quality peer-reviewed journal.
(pichardkostuch2023theuniversalsua5tsac pages 1-3): Adeline Pichard-Kostuch, Violette Da Cunha, Jacques Oberto, Ludovic Sauguet, and Tamara Basta. The universal sua5/tsac family evolved different mechanisms for the synthesis of a key trna modification. Frontiers in Microbiology, Jun 2023. URL: https://doi.org/10.3389/fmicb.2023.1204045, doi:10.3389/fmicb.2023.1204045. This article has 9 citations and is from a peer-reviewed journal.
(pichardkostuch2023theuniversalsua5tsac pages 3-4): Adeline Pichard-Kostuch, Violette Da Cunha, Jacques Oberto, Ludovic Sauguet, and Tamara Basta. The universal sua5/tsac family evolved different mechanisms for the synthesis of a key trna modification. Frontiers in Microbiology, Jun 2023. URL: https://doi.org/10.3389/fmicb.2023.1204045, doi:10.3389/fmicb.2023.1204045. This article has 9 citations and is from a peer-reviewed journal.
(lauhon2012mechanismofn6threonylcarbamoyladenonsine pages 1-2): Charles T. Lauhon. Mechanism of n6-threonylcarbamoyladenonsine (t(6)a) biosynthesis: isolation and characterization of the intermediate threonylcarbamoyl-amp. Biochemistry, 51 44:8950-63, Oct 2012. URL: https://doi.org/10.1021/bi301233d, doi:10.1021/bi301233d. This article has 85 citations and is from a peer-reviewed journal.
(pichardkostuch2023theuniversalsua5tsac media 03316335): Adeline Pichard-Kostuch, Violette Da Cunha, Jacques Oberto, Ludovic Sauguet, and Tamara Basta. The universal sua5/tsac family evolved different mechanisms for the synthesis of a key trna modification. Frontiers in Microbiology, Jun 2023. URL: https://doi.org/10.3389/fmicb.2023.1204045, doi:10.3389/fmicb.2023.1204045. This article has 9 citations and is from a peer-reviewed journal.
(choi2024sua5catalyzinguniversal pages 1-2): Yeseul Choi, Hana Hyeon, Kangseok Lee, and Yong-Sun Bahn. Sua5 catalyzing universal t 6 a trna modification is responsible for multifaceted functions of the keops complex in cryptococcus neoformans. mSphere, Jan 2024. URL: https://doi.org/10.1128/msphere.00557-23, doi:10.1128/msphere.00557-23. This article has 6 citations and is from a peer-reviewed journal.
(pichardkostuch2023theuniversalsua5tsac pages 10-12): Adeline Pichard-Kostuch, Violette Da Cunha, Jacques Oberto, Ludovic Sauguet, and Tamara Basta. The universal sua5/tsac family evolved different mechanisms for the synthesis of a key trna modification. Frontiers in Microbiology, Jun 2023. URL: https://doi.org/10.3389/fmicb.2023.1204045, doi:10.3389/fmicb.2023.1204045. This article has 9 citations and is from a peer-reviewed journal.
(guo2024newinsightsinto pages 1-2): Haiyong Guo, Ting Lei, Junshu Yang, Yue Wang, Yifan Wang, and Yinduo Ji. New insights into the biological functions of essential tsab/yeaz protein in staphylococcus aureus. Antibiotics, 13:393, Apr 2024. URL: https://doi.org/10.3390/antibiotics13050393, doi:10.3390/antibiotics13050393. This article has 2 citations.
(guo2024newinsightsinto pages 9-11): Haiyong Guo, Ting Lei, Junshu Yang, Yue Wang, Yifan Wang, and Yinduo Ji. New insights into the biological functions of essential tsab/yeaz protein in staphylococcus aureus. Antibiotics, 13:393, Apr 2024. URL: https://doi.org/10.3390/antibiotics13050393, doi:10.3390/antibiotics13050393. This article has 2 citations.
(guo2024newinsightsinto pages 11-12): Haiyong Guo, Ting Lei, Junshu Yang, Yue Wang, Yifan Wang, and Yinduo Ji. New insights into the biological functions of essential tsab/yeaz protein in staphylococcus aureus. Antibiotics, 13:393, Apr 2024. URL: https://doi.org/10.3390/antibiotics13050393, doi:10.3390/antibiotics13050393. This article has 2 citations.
(lauhon2012mechanismofn6threonylcarbamoyladenonsine pages 11-12): Charles T. Lauhon. Mechanism of n6-threonylcarbamoyladenonsine (t(6)a) biosynthesis: isolation and characterization of the intermediate threonylcarbamoyl-amp. Biochemistry, 51 44:8950-63, Oct 2012. URL: https://doi.org/10.1021/bi301233d, doi:10.1021/bi301233d. This article has 85 citations and is from a peer-reviewed journal.
(lauhon2012mechanismofn6threonylcarbamoyladenonsine pages 8-9): Charles T. Lauhon. Mechanism of n6-threonylcarbamoyladenonsine (t(6)a) biosynthesis: isolation and characterization of the intermediate threonylcarbamoyl-amp. Biochemistry, 51 44:8950-63, Oct 2012. URL: https://doi.org/10.1021/bi301233d, doi:10.1021/bi301233d. This article has 85 citations and is from a peer-reviewed journal.
(lauhon2012mechanismofn6threonylcarbamoyladenonsine pages 9-11): Charles T. Lauhon. Mechanism of n6-threonylcarbamoyladenonsine (t(6)a) biosynthesis: isolation and characterization of the intermediate threonylcarbamoyl-amp. Biochemistry, 51 44:8950-63, Oct 2012. URL: https://doi.org/10.1021/bi301233d, doi:10.1021/bi301233d. This article has 85 citations and is from a peer-reviewed journal.
(thiaville2014diversityofthe pages 2-3): Patrick C Thiaville, Dirk Iwata-Reuyl, and Valérie de Crécy-Lagard. Diversity of the biosynthesis pathway for threonylcarbamoyladenosine (t6a), a universal modification of trna. RNA Biology, 11:1529-1539, Dec 2014. URL: https://doi.org/10.4161/15476286.2014.992277, doi:10.4161/15476286.2014.992277. This article has 114 citations and is from a peer-reviewed journal.
(pichardkostuch2023theuniversalsua5tsac media ef9a6773): Adeline Pichard-Kostuch, Violette Da Cunha, Jacques Oberto, Ludovic Sauguet, and Tamara Basta. The universal sua5/tsac family evolved different mechanisms for the synthesis of a key trna modification. Frontiers in Microbiology, Jun 2023. URL: https://doi.org/10.3389/fmicb.2023.1204045, doi:10.3389/fmicb.2023.1204045. This article has 9 citations and is from a peer-reviewed journal.
(harris2015nmrbasedstructuralanalysis pages 8-10): Kimberly A. Harris, Benjamin G. Bobay, Kathryn L. Sarachan, Alexis F. Sims, Yann Bilbille, Christopher Deutsch, Dirk Iwata-Reuyl, and Paul F. Agris. Nmr-based structural analysis of threonylcarbamoyl-amp synthase and its substrate interactions. Journal of Biological Chemistry, 290:20032-20043, Aug 2015. URL: https://doi.org/10.1074/jbc.m114.631242, doi:10.1074/jbc.m114.631242. This article has 16 citations and is from a domain leading peer-reviewed journal.
id: P0AFR4
gene_symbol: yciO
product_type: PROTEIN
status: COMPLETE
taxon:
id: NCBITaxon:83333
label: Escherichia coli (strain K12)
description: >-
YciO is an uncharacterized member of the PF01300 (TsaC/Sua5/YciO/YrdC) family in E. coli.
It is a PARALOG of TsaC (L-threonylcarbamoyladenylate synthase, EC 2.7.7.87), but extensive
evidence demonstrates that YciO does NOT perform the same biological function as TsaC.
While YciO shows very weak L-threonylcarbamoyladenylate synthase activity in vitro
(0.14 nM/min TC-AMP production, PMID:37963869), this is more than 4 orders of magnitude
weaker than TsaC (2.8 uM/min) under similar conditions (PMID:40703034). The canonical
KRSN tetrad (KxR...SxN) required for TC-AMP synthesis is replaced by KxL...SxM in YciO,
consistent with altered or lost catalytic activity. In vivo experiments have shown that
TsaC is essential even when yciO is present or overexpressed, indicating YciO cannot
substitute for TsaC function. The weak in vitro activity is likely residual
ancestral/promiscuous activity, not the biological function. YciO possesses a large conserved
positively charged surface (absent in TsaC) predicted to interact with RNA (PMID:40703034).
Genomic context analysis shows yciO genes frequently colocalize with rnm genes encoding
RNase AM, a 5-to-3 exonuclease that matures the 5-prime ends of rRNAs. The true biological
function of YciO is unknown but likely relates to rRNA metabolism rather than tRNA modification.
This gene is a textbook case of how paralog confusion and uncritical acceptance of in vitro
promiscuous activity can lead to incorrect functional annotations.
existing_annotations:
- term:
id: GO:0005829
label: cytosol
evidence_type: IBA
original_reference_id: GO_REF:0000033
review:
summary: >-
IBA annotation for cytosol localization based on phylogenetic inference (PANTHER).
Consistent with experimental IDA evidence from two independent proteomics studies
(PMID:15911532, PMID:18304323) that identified YciO in the cytosolic fraction of
E. coli K-12.
action: ACCEPT
reason: >-
Cytosolic localization is well-supported by both phylogenetic inference and direct
experimental evidence from mass spectrometry-based proteomics. Two independent studies
identified YciO as a cytosolic protein.
supported_by:
- reference_id: PMID:15911532
supporting_text: >-
Here we describe a proteomic analysis of Escherichia coli in which 3,199 protein
forms were detected, and of those 2,160 were annotated and assigned to the
cytosol, periplasm, inner membrane, and outer membrane by biochemical
fractionation followed by two-dimensional gel electrophoresis and tandem mass
spectrometry.
- reference_id: PMID:18304323
supporting_text: >-
Here, we describe an experimental scheme to maximize the coverage of
proteins identified by mass spectrometry of a complex biological sample.
- term:
id: GO:0003725
label: double-stranded RNA binding
evidence_type: IEA
original_reference_id: GO_REF:0000002
review:
summary: >-
IEA annotation based on InterPro domain IPR006070 (Sua5-like domain) mapping to
dsRNA binding. This is a computational prediction from InterPro. While YciO does
possess a large conserved positively charged surface predicted to interact with RNA
(PMID:40703034), the specific annotation as dsRNA binding is not experimentally
validated and the InterPro mapping may be overly specific. The positively charged
surface is a feature distinguishing YciO from TsaC, suggesting RNA interaction
is plausible, but the specific type of RNA (dsRNA vs rRNA) is uncertain.
action: UNDECIDED
reason: >-
The InterPro-based prediction of dsRNA binding is plausible given the large
positively charged surface unique to YciO proteins (PMID:40703034), but there is no
direct experimental evidence for dsRNA binding specifically. The genomic context
(colocalization with rnm genes encoding RNase AM, a rRNA maturation enzyme)
suggests rRNA interaction may be more likely. Without experimental evidence, it is
difficult to determine whether dsRNA binding or a more specific RNA binding term
is appropriate. The annotation should be revisited when experimental data on YciO
RNA binding become available.
supported_by:
- reference_id: PMID:40703034
supporting_text: >-
the structure of YciO exhibits a large positively charged surface predicted
to interact with RNA
- reference_id: PMID:40703034
supporting_text: >-
genes are colocalized with rnm genes (Fig. 7), which encode the recently
characterized RNase AM, a 5′ to 3′ exonuclease that matures the 5′ end of
all 3 ribosomal RNAs in E. coli
- term:
id: GO:0061710
label: L-threonylcarbamoyladenylate synthase activity
evidence_type: IEA
original_reference_id: GO_REF:0000003
review:
summary: >-
IEA annotation based on EC number mapping (EC:2.7.7.87). This EC number was assigned
to YciO in UniProt based on the Kim et al. 2023 deep learning prediction and in vitro
assay (PMID:37963869). However, de Crecy-Lagard et al. 2025 (PMID:40703034) demonstrated
that this is a classic case of paralog incorrect (PLI) annotation. YciO is a paralog of
TsaC but does not perform the same biological function. The in vitro activity is 4 orders
of magnitude weaker than TsaC and represents residual ancestral/promiscuous activity.
In vivo experiments show TsaC is essential even with yciO present. The EC2GO mapping
propagates this incorrect EC assignment.
action: REMOVE
reason: >-
This annotation propagates through EC2GO from the incorrect EC:2.7.7.87 assignment
to YciO. De Crecy-Lagard et al. 2025 (PMID:40703034) provide compelling evidence that
YciO does NOT function as an L-threonylcarbamoyladenylate synthase in vivo. The weak
in vitro activity (0.14 nM/min vs 2.8 uM/min for TsaC) is residual ancestral activity,
not the biological function. In vivo, TsaC is essential even when yciO is overexpressed.
The EC number 2.7.7.87 prediction was given a confidence score of 0 (refuted) by expert
curation in the de Crecy-Lagard study.
additional_reference_ids:
- PMID:40703034
supported_by:
- reference_id: PMID:40703034
supporting_text: >-
YciO does not perform the same function as TsaC/Susa5 in vivo experiments
- reference_id: PMID:40703034
supporting_text: >-
the activity reported (0.14 nM/min TC-AMP production rate) for E. coli
YciO is more than 4 orders of magnitude weaker than that of E. coli
TsaC (2.8 μM/min) at the same enzyme concentration and similar reaction
conditions
- reference_id: PMID:40703034
supporting_text: >-
the functional puzzle is far from being solved for proteins of the YciO subgroup,
and even if the existing data suggest a role in RNA metabolism, it cannot be the
same as TsaC, and the EC number 2.7.7.87 prediction was given a CS of 0
- term:
id: GO:0061710
label: L-threonylcarbamoyladenylate synthase activity
evidence_type: IDA
original_reference_id: PMID:37963869
review:
summary: >-
IDA annotation based on Kim et al. 2023 (PMID:37963869), which used DeepECTransformer
to predict EC 2.7.7.87 for YciO and validated it with an in vitro enzyme assay
showing a specific activity of 0.0705 U/mg. However, de Crecy-Lagard et al. 2025
(PMID:40703034) demonstrated this is a paralog incorrect (PLI) prediction. The measured
activity (0.14 nM/min TC-AMP) is more than 4 orders of magnitude weaker than genuine
TsaC activity (2.8 uM/min). This weak activity represents residual ancestral/promiscuous
catalytic activity from shared evolutionary origin, not the biological function. In vivo
experiments show YciO cannot substitute for TsaC. Models of enzyme evolution predict
that paralogs retain promiscuous ancestral activities detectable in vitro. The IDA
evidence code is technically correct for in vitro detection, but the annotation
misrepresents the biological function of YciO.
action: REMOVE
reason: >-
While Kim et al. 2023 (PMID:37963869) did detect L-threonylcarbamoyladenylate synthase
activity in vitro, de Crecy-Lagard et al. 2025 (PMID:40703034) conclusively demonstrate
this is promiscuous/residual ancestral activity, not the biological function. The activity
is 4 orders of magnitude weaker than TsaC. In vivo, TsaC is essential even with YciO
present or overexpressed. The Kim et al. study used the in vitro assay to validate a
deep learning prediction without considering the in vivo context, genomic evidence, or
the distinction between promiscuous and biological activity. As stated in PMID:40703034:
in vitro activity alone is not sufficient to validate protein function in vivo. This
annotation should be removed as it represents an over-annotation based on promiscuous
activity of a paralog.
additional_reference_ids:
- PMID:40703034
supported_by:
- reference_id: PMID:37963869
supporting_text: >-
In the case of YciO, which was previously annotated to belong to the SUA5 family,
DeepECtransformer predicted its EC number to be EC:2.7.7.87
(L-threonylcarbamoyladenylate synthase) with the prediction score of 0.9108.
The specific activity of YciO was measured to be 0.0705 U mg-1
- reference_id: PMID:40703034
supporting_text: >-
the activity reported (0.14 nM/min TC-AMP production rate) for E. coli
YciO is more than 4 orders of magnitude weaker than that of E. coli
TsaC (2.8 μM/min) at the same enzyme concentration and similar reaction
conditions ... consistent with the possibility of a missing partner or a
different biological substrate for YciO
- reference_id: PMID:40703034
supporting_text: >-
YciO does not perform the same function as TsaC/Susa5 in vivo experiments
- reference_id: PMID:40703034
supporting_text: >-
TsaC is an enzyme predicted to have been present in the Last Universal Common
Ancestor ... YciO is a likely paralog of TsaC (Fig. 6), and as such, it is
likely to have residual ancestral catalytic activity that can be detected in
vitro
- reference_id: PMID:40703034
supporting_text: >-
in vitro activity alone is not sufficient to validate the function of a
protein in vivo
- reference_id: file:ECOLI/yciO/yciO-deep-research-falcon.md
supporting_text: Falcon deep research independently confirms YciO is not part
of the canonical t6A pathway. Multiple sources (Harris 2015, Thiaville 2014,
Carvalho 2017, Pichard-Kostuch 2023) establish YciO as a divergent paralog
lacking the conserved KRSN tetrad required for TC-AMP synthesis.
- term:
id: GO:0005829
label: cytosol
evidence_type: IDA
original_reference_id: PMID:15911532
review:
summary: >-
IDA annotation for cytosol localization from Lopez-Campistrous et al. 2005
(PMID:15911532), a large-scale proteomics study of E. coli K-12 that assigned
proteins to subcellular compartments by biochemical fractionation followed by
2D gel electrophoresis and tandem mass spectrometry.
action: ACCEPT
reason: >-
Direct experimental evidence from proteomics-based subcellular fractionation.
The study identified 2,160 protein forms and assigned them to cytosol, periplasm,
inner membrane, and outer membrane. YciO is a soluble cytoplasmic protein
consistent with this localization.
supported_by:
- reference_id: PMID:15911532
supporting_text: >-
Here we describe a proteomic analysis of Escherichia coli in which 3,199 protein
forms were detected, and of those 2,160 were annotated and assigned to the
cytosol, periplasm, inner membrane, and outer membrane by biochemical
fractionation followed by two-dimensional gel electrophoresis and tandem mass
spectrometry.
- term:
id: GO:0005829
label: cytosol
evidence_type: IDA
original_reference_id: PMID:18304323
review:
summary: >-
IDA annotation for cytosol localization from Ishihama et al. 2008 (PMID:18304323),
a comprehensive protein abundance profiling study of the E. coli cytosol using
mass spectrometry.
action: ACCEPT
reason: >-
Independent experimental confirmation of cytosolic localization from a second
large-scale proteomics study. Consistent with the IDA annotation from PMID:15911532
and the IBA annotation.
supported_by:
- reference_id: PMID:18304323
supporting_text: >-
Here, we describe an experimental scheme to maximize the coverage of
proteins identified by mass spectrometry of a complex biological sample.
- term:
id: GO:0003723
label: RNA binding
evidence_type: ISS
original_reference_id: PMID:40703034
review:
summary: >-
NEW annotation. YciO possesses a large conserved positively charged surface
(comprising ~6% of total molecular surface area) that is present and conserved in
all YciO family members but absent in TsaC proteins (PMID:40703034). This surface
is predicted to interact with RNA. Genomic context shows yciO genes colocalize with
rnm genes (encoding RNase AM, a rRNA maturation enzyme), suggesting involvement in
rRNA metabolism.
action: NEW
reason: >-
De Crecy-Lagard et al. 2025 (PMID:40703034) provide structural and genomic context
evidence that YciO has a conserved positively charged surface predicted to bind RNA.
While the specific RNA target has not been experimentally determined, general RNA
binding is well supported by structural analysis. This is proposed as a more
appropriate molecular function annotation than the incorrect L-threonylcarbamoyladenylate
synthase activity.
additional_reference_ids:
- PMID:40703034
supported_by:
- reference_id: PMID:40703034
supporting_text: >-
the structure of YciO exhibits a large positively charged surface predicted
to interact with RNA
- reference_id: PMID:40703034
supporting_text: >-
This large positively charged surface is conserved in YciO proteins and is
absent in TsaC proteins
references:
- id: GO_REF:0000002
title: Gene Ontology annotation through association of InterPro records with GO terms
findings: []
- id: GO_REF:0000003
title: Gene Ontology annotation based on Enzyme Commission mapping
findings: []
- id: GO_REF:0000033
title: Annotation inferences using phylogenetic trees
findings: []
- id: PMID:15911532
title: Localization, annotation, and comparison of the Escherichia coli K-12 proteome
under two states of growth.
findings:
- statement: YciO was identified as a cytosolic protein by biochemical fractionation
and mass spectrometry in E. coli K-12
supporting_text: >-
Here we describe a proteomic analysis of Escherichia coli in which 3,199 protein
forms were detected, and of those 2,160 were annotated and assigned to the
cytosol, periplasm, inner membrane, and outer membrane by biochemical
fractionation followed by two-dimensional gel electrophoresis and tandem mass
spectrometry.
- id: PMID:18304323
title: Protein abundance profiling of the Escherichia coli cytosol.
findings:
- statement: YciO was detected in the E. coli cytosolic fraction by mass spectrometry-based
protein abundance profiling
supporting_text: >-
Here, we describe an experimental scheme to maximize the coverage of
proteins identified by mass spectrometry of a complex biological sample.
- id: PMID:37963869
title: Functional annotation of enzyme-encoding genes using deep learning with transformer
layers.
findings:
- statement: DeepECTransformer predicted YciO has EC 2.7.7.87 (L-threonylcarbamoyladenylate
synthase) activity based on sequence features matching TIGR00057 family
supporting_text: >-
In the case of YciO, which was previously annotated to belong to the SUA5 family,
DeepECtransformer predicted its EC number to be EC:2.7.7.87
(L-threonylcarbamoyladenylate synthase) with the prediction score of 0.9108.
- statement: In vitro enzyme assay detected a specific activity of 0.0705 U/mg for
L-threonylcarbamoyladenylate synthase activity, but this was not validated in vivo
supporting_text: >-
The specific activity of YciO was measured to be 0.0705 U mg-1
- id: DOI:10.4161/15476286.2014.992277
title: Diversity of the biosynthesis pathway for threonylcarbamoyladenosine (t6A),
a universal modification of tRNA.
findings:
- statement: YciO is described as a paralog lacking the conserved KRSN tetrad
(KxR...SxN motif) found in functional TsaC/Sua5 proteins, and does not have
the same function. YciO is present in ~54% of 9,176 bacterial genomes analyzed.
supporting_text: YciO is described as a paralog lacking the conserved KRSN
tetrad and not having the same function
- id: DOI:10.1074/jbc.m114.631242
title: NMR-based structural analysis of threonylcarbamoyl-AMP synthase and its
substrate interactions.
findings:
- statement: Structural study of E. coli TsaC/YrdC explicitly states that YciO
is not associated with the t6A37 pathway, despite structural similarity.
supporting_text: E. coli YciO has a solved structure but is not associated
with the t6A37 pathway
- id: DOI:10.1016/j.ympev.2016.12.033
title: Slr0006-like proteins - a TsaC/TsaC2/YciO subfamily exclusive to
cyanobacteria.
findings:
- statement: Phylogenetic/motif analysis emphasizes YciO's divergence from TsaC,
nonessentiality, and inability to compensate for TsaC loss. The canonical
KxR...SxN motif is replaced by KxL...SxM in E. coli YciO.
supporting_text: YciO's divergence and nonessentiality, and inability to
compensate for TsaC loss, consistent with functional separation
- id: DOI:10.3389/fmicb.2023.1204045
title: The universal Sua5/TsaC family and the associated modifying enzymes of
the t6A tRNA modification pathway.
findings:
- statement: Modern phylogenomic synthesis confirms Sua5 as ancestral form, TsaC
as derived, and YciO as an inactive TsaC paralog in bacterial lineages. The
family is universally conserved and essential for t6A biogenesis.
supporting_text: YciO is discussed as an inactive TsaC paralog in bacterial
lineages
- id: PMID:40703034
title: Limitations of current machine learning models in predicting enzymatic functions
for uncharacterized proteins.
findings:
- statement: YciO is a paralog of TsaC that does NOT perform the same biological function;
the in vitro L-threonylcarbamoyladenylate synthase activity (0.14 nM/min) is more
than 4 orders of magnitude weaker than TsaC (2.8 uM/min) and represents residual
ancestral/promiscuous activity
supporting_text: >-
the activity reported (0.14 nM/min TC-AMP production rate) for E. coli
YciO is more than 4 orders of magnitude weaker than that of E. coli
TsaC (2.8 μM/min) at the same enzyme concentration and similar reaction
conditions
- statement: YciO possesses a large conserved positively charged surface (absent in TsaC)
predicted to interact with RNA, and yciO genes colocalize with rnm genes encoding
RNase AM (rRNA maturation enzyme)
supporting_text: >-
the structure of YciO exhibits a large positively charged surface predicted
to interact with RNA
- statement: In vivo experiments show YciO cannot substitute for TsaC function; the
DeepECTransformer EC 2.7.7.87 prediction was given a confidence score of 0 (refuted)
by expert curation
supporting_text: >-
YciO does not perform the same function as TsaC/Susa5 in vivo experiments
- statement: This is a textbook example of paralog incorrect (PLI) annotation error,
where in vitro promiscuous activity does not reflect biological function
supporting_text: >-
in vitro activity alone is not sufficient to validate the function of a
protein in vivo
core_functions:
- description: >-
YciO is a protein of unknown biological function that likely participates in rRNA
metabolism based on genomic context (colocalization with rnm/RNase AM genes) and
structural features (conserved positively charged surface predicted to bind RNA).
It is NOT an L-threonylcarbamoyladenylate synthase despite weak in vitro activity,
which represents residual ancestral activity from its evolutionary relationship to
TsaC.
molecular_function:
id: GO:0003723
label: RNA binding
supported_by:
- reference_id: PMID:40703034
supporting_text: >-
the structure of YciO exhibits a large positively charged surface predicted
to interact with RNA ... This large positively charged surface is conserved
in YciO proteins and is absent in TsaC proteins
suggested_questions:
- question: What is the actual RNA substrate of YciO?
- question: Does YciO physically interact with RNase AM (Rnm) or other rRNA processing factors?
- question: Should the UniProt record for YciO (P0AFR4) be corrected to remove the EC 2.7.7.87 assignment?
suggested_experiments:
- description: >-
RNA co-immunoprecipitation or CLIP-seq to identify RNA binding partners of YciO.
YciO has a conserved positively charged surface predicted to bind RNA. Identifying
the actual RNA targets (rRNA, tRNA, mRNA, or other) would resolve the functional puzzle.
hypothesis: YciO binds rRNA based on its conserved positively charged surface and genomic
colocalization with rnm (RNase AM) genes.
- description: >-
In vivo complementation tests with yciO deletion in combination with rnm deletion.
Genomic context suggests functional coupling between YciO and RNase AM. A synthetic
interaction would support the hypothesis that YciO participates in rRNA maturation.
hypothesis: YciO and RNase AM (Rnm) function in the same rRNA maturation pathway,
and double deletion will show a synthetic phenotype.
- description: >-
Electrophoretic mobility shift assays (EMSAs) with various RNA substrates including
rRNA fragments, tRNA, and dsRNA to narrow down the RNA binding specificity suggested
by the positively charged surface.
hypothesis: YciO preferentially binds rRNA over other RNA species.