Biology subjects

Lee, Y.-J.

Publications and source records attributed to Lee, Y.-J..

At least 19 recordsLinked to original sources

bioRxiv2026

Unlocking the secrets of paired terminal genomic repeats utilised by the Burkholderia jumbo phage Matilda

Bacteriophages (phages) are viruses that infect bacteria. Jumbo phages have genomes exceeding 220 kb, which can support greater functional independence and complexity. This work identified vB_BceM_Matilda ({phi}Matilda), a novel Burkholderia-targeting jumbo phage. Before injecting its genome into the host bacterium, {phi}Matilda binds to the bacterial flagellum, the rotation of which is required to drive a productive phage infection. Its linear 267 kb dsDNA genome has a previously undescribed tripartite architecture, with bidirectional terminal elements of 1.3 kb and 25 kb, each flanked by distinct inverted repeats of 1.5 kb and 18 kb, respectively. These architectural elements bookend the genome. Transcriptomic and proteomic analyses tracked the phage infection process and confirmed that genes within these terminal elements are transcribed and translated during infection. The presence of bidirectional terminal sequences in related jumbo phages suggests that the tripartite genome architecture may be conserved and functionally important. This work represents the first description of this genome architecture in any organism, expanding our understanding of genome organisation and raising new questions about the replication, packaging, and evolution of jumbo phage genomes.

microbiology↗

bioRxiv2026

ATML1-GIR1-TPL/TPR transcriptional repression module controls glucosinolates and giant cells in Arabidopsis thaliana sepals

Glucosinolates (GSLs) are sulfur- and nitrogen-containing secondary metabolites that serve as defense compounds in Arabidopsis and other members of the Brassicales. Although the enzymatic pathway that produces GSLs is well-studied, the upstream mechanisms that control their tissue-specific synthesis are poorly understood. We identified a novel repression module that transcriptionally regulates GSL levels in sepals, the modified leaves that protect reproductive tissues within the floral bud. GLABRA2 (GL2) INTERACTING REPRESSOR1 (GIR1) interacts directly with Arabidopsis thaliana MERISTEM LAYER1 (ATML1), an HD-Zip IV transcription factor known to be required for giant cell formation in the sepal epidermis. This interaction requires a predicted Zn finger of GIR1 and the C-terminal START adjacent domain (STAD) of ATML1. The gir1 loss-of-function mutants exhibit excess giant cells, in contrast to atml1 mutants which display fewer giant cells, supporting the role of GIR1 as a negative regulator of ATML1. We confirmed that GIR1 interacts with TOPLESS (TPL) and TOPLESS-RELATED (TPR) corepressors, and coimmunoprecipitation demonstrated that GIR1 acts as an adaptor protein connecting ATML1 and TPL/TPR. RNA sequencing revealed that numerous genes involved in GSL biosynthesis, including the key transcriptional regulator MYB29, are upregulated in gir1 mutants. Consistent with the transcriptomic data, chemical analysis revealed that gir1 mutants display elevated GSL levels in sepals. Mass spectrometry imaging confirmed high GSL accumulation in gir1 sepals compared to wild type and atml1. Overall, our findings uncover a previously unrecognized link between cell expansion and GSL metabolism, suggesting strategies for engineering plants with cell-type specific GSL profiles. Significance StatementPlants belonging to the order Brassicales produce sulfur-containing glucosinolate (GSL) metabolites that serve in defense against herbivory. In cruciferous vegetables such as broccoli and kale, these compounds contribute to their unique flavors and health-promoting attributes. In agriculturally important oilseed crops, they affect the palatability of animal feeds. Here, we identified a novel transcriptional regulatory module that controls GSL biosynthesis in the epidermis of the sepal, the floral organ that protects the reproductive tissues. This regulatory module also controls cell expansion of specific cell types in the sepal, demonstrating a surprising connection between cell growth and a chemical defense pathway in plants. Our results suggest strategies for engineering crops with tissue-specific GSL profiles to fit agronomic needs.

plant biology↗

bioRxiv2026

Age-dependent pathogenicity of two severe fever with thrombocytopenia syndrome viruses in a ferret model

SFTSV is an emerging tick-borne pathogen associated with high case fatality rates, particularly in elderly patients. While severe pathogenicity has been reported in aged ferrets, lethal or clinically significant infection in younger animals and genotype-dependent differences in pathogenicity remain insufficiently defined. In this study, we established a ferret infection model using two Korean clinical isolates representing genotypes B and F and systematically compared disease progression between one-year-old and three-year-old ferrets. Three-year-old ferrets exhibited rapid fever onset, marked body weight loss, early clinical deterioration, severe thrombocytopenia and leukopenia, significant elevations in AST and ALT levels, and earlier peak viremia with higher tissue viral loads, indicating impaired early viral control and accelerated systemic dissemination. Notably, one-year-old ferrets also developed measurable pathogenic manifestations, including febrile responses, progressive weight loss, detectable viremia, and multiorgan viral distribution, although disease progression was delayed and less severe compared with older animals. Within the same age group, differences in pathogenicity between genotypes B and F were limited. These findings demonstrate that host age is a major determinant of SFTSV disease severity and support the use of an age-stratified ferret model for preclinical evaluation of vaccines and antiviral therapeutics. ImportanceSFTS is an emerging tick-borne disease that can cause high fever, thrombocytopenia, and multi-organ failure, with particularly severe outcomes in older adults. Currently, no approved vaccines or specific antiviral treatments are available. Reliable animal models that recapitulate human disease are therefore essential for the development of effective countermeasures. Ferrets have recently been proposed as a useful model for SFTS, especially in aged animals, but the susceptibility of younger ferrets and the impact of viral strain differences remain unclear. Here we show that host age strongly determines disease severity in ferrets infected with two genetically distinct SFTS virus strains, establishing a flexible animal model for evaluating vaccines and antiviral therapies.

microbiology↗

bioRxiv2026

A methylome-derived m6-dAMP trigger assembles a PUA-Cal-HAD immune filament that depletes dNTPs to abort phage infection

Bacteria must distinguish phage attack from normal homeostatic processes, yet the danger signals that trigger many defence systems remain unknown. Here, we show that a PUA-Calcineurin-CE-HAD module from Escherichia coli ECOR28 confers broad anti-phage protection by binding Dam-methylated deoxyadenosine monophosphate (m6-dAMP) generated during phage-induced chromosome degradation. Ligand binding converts a preassembled PUA-Calcineurin-CE hexamer loaded with six HAD phosphatases into a polymerising filament. The filament acts as a high-flux dNTP sink through a two-enzyme cascade: HAD first dephosphorylates dATP to dADP, and Calcineurin-CE then converts dADP to dAMP. dNTP collapse halts phage replication and enforces abortive infection. Multiple mobile-element DNA mimic proteins block filament assembly, revealing a direct phage counter-defence. More broadly, our findings extend a conserved, cross-kingdom paradigm of immune filament assembly to nucleotide-depletion antiviral defence and suggest modified-nucleotide sensing by related PUA-Calcineurin-CE modules as a widespread, underappreciated bacterial strategy.

microbiology↗

bioRxiv2025

Antiviral Defence is a Conserved Function of Diverse DNA Glycosylases

Bacteria are frequently attacked by viruses, known as phages, and rely on diverse defence systems like restriction endonucleases and CRISPR-Cas to survive. While phages can evade these defences by covalently modifying their DNA, these non-canonical nucleobases create a strong selective pressure for host proteins that can recognize and exploit them. Here, using a structure-guided discovery approach, we identify widespread families of DNA glycosylases that protect bacteria against phages that incorporate modified guanine bases into their DNA. Despite high sequence variation, these enzymes share a conserved glycosylase fold and occur across bacterial lineages. We also uncover a distinct glycosylase superfamily that defends against phages with thymidine modifications, showing that glycosylases have repeatedly evolved as antiviral defences. Together, these findings reveal DNA glycosylases as versatile effectors of bacterial immunity and underscore structure-guided discovery as a powerful strategy for uncovering hidden layers of antiviral defence.

microbiology↗

bioRxiv2025

Keratin degradation reflects a starvation survival strategy in Fervidobacterium islandicum AW-1

Keratin is a highly cross-linked, disulfide-rich protein that resists proteolysis, which poses a major challenge for microbial degradation. Here, we show that Fervidobacterium islandicum AW-1 initiates a starvation-induced keratinolytic program involving membrane-associated proteases and redox-mediated sulfitolysis. Multi-omics integration reveals that nutrient limitation triggers global metabolic reprogramming, promoting sulfur assimilation, biofilm formation, and chemotaxis-linked persister-like adaptation. Substrate-specific transcriptomics identified a temporally regulated protease repertoire tightly coordinated with sulfitolytic activity, facilitating efficient feather decomposition under starvation. Protein-protein interaction networks uncovered stress-responsive transcriptional regulators that govern this process. Time-resolved gene expression analysis and metabolomic profiling further revealed that cyclic-di-GMP signaling, stringent response, and flagella assembly mediate transitions between motility and sessile growth, contributing to surface colonization and persistence. Together, our findings establish a starvation-responsive survival mechanism that couples keratin degradation to stress adaptation in extreme environments, offering insights into microbial persistence and potential strategies for keratin valorization.

microbiology↗

bioRxiv2025

Biosynthesis of glycosylated 5-hydroxycytosine in the DNA of diverse viruses

Characterizing viral strategies for circumventing cellular defenses is critical to understanding the biology and therapeutic application of bacteriophages as antimicrobials. Many bacteriophages synthesize complex modifications (i.e., hypermodifications) to the nucleobases of their virion DNA in order to circumvent the endonuclease-based defenses of their hosts. To date, most hypermodified pyrimidines in viruses are synthesized via group transfer to a pre-existing hydroxyl moiety in DNA during the later stages of lytic development leading up to packaging of the viral chromosome into the capsid. Typically, these occur at hydroxymethylcytosine or hydroxymethyluracil. We find mono- and poly-arabinosylated cytidines completely replacing canonical cytidine in the native DNA of the Escherichia phage RB69 and Acinetobacter baumannii phage DLP3. In both cases, an arabinosyl moiety is connected directly to the pyrimidine through an ether linkage with the bridging oxygen originating from 5-hydroxycytosine (5hoC) in DNA. The 5hoC is synthesized as a mononucleotide by a virally encoded thymidylate synthase homolog. We find diverse virally encoded thymidylate synthase homologs, including the Rhizobium phage RL38J1, are capable of producing 5hoC suggesting this base modification is a starting point for glycodiverse cytosine derivatives. Characterizing these cytosine derivatives adds to our understanding of viral anti-defense mechanisms. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=81 SRC="FIGDIR/small/659550v1_ufig1.gif" ALT="Figure 1"> View larger version (19K): org.highwire.dtl.DTLVardef@fcc165org.highwire.dtl.DTLVardef@1fced2aorg.highwire.dtl.DTLVardef@369ceaorg.highwire.dtl.DTLVardef@177df60_HPS_FORMAT_FIGEXP M_FIG C_FIG

microbiology↗

bioRxiv2025

Developmental system drift in dorsoventral patterning is linked to transitions to autonomous development in Annelida

The Bone Morphogenetic Protein (BMP) pathway is the ancestral signalling system defining the dorsoventral axis in bilaterally symmetrical animals. However, Spiralia, a large bilaterian clade including molluscs and annelids, uses the Fibroblast Growth Factor pathway and ERK1/2 as the ancestral cue to establish their posterodorsal side. How this profound change in axial patterning evolved and what it implied for BMPs developmental role remains elusive. Here, we studied four annelid species and combined disruption of the BMP and Activin/Nodal pathways with transcriptomics and blastomere deletions to demonstrate that BMP is ancestrally downstream of ERK1/2 and promotes dorsoventral development in Spiralia. Importantly, this signalling hierarchy is lost in annelids that secondarily transitioned into a maternally controlled, autonomous development. While some, like Capitella teleta, use Activin/Nodal, Platynereis dumerilii relies on BMP to establish dorsoventral polarity only in the head. Unexpectedly, this divergence in upstream axial regulators implied extensive rewiring of downstream targets, as inferred by comparing C. teleta and Owenia fusiformis. Our data clarify the ancestral axial role for BMP in Spiralia, unveiling a potential causal link between parallel shifts to autonomous cell-fate specification in early development and the emergence of developmental system drift, a pervasive yet poorly understood phenomenon in animal embryogenesis.

developmental biology↗

bioRxiv2025

END nucleases: Antiphage defense systems targeting multiple hypermodified phage genomes

Prokaryotes carry clusters of phage defense systems in "defense islands" that have been extensively exploited bioinformatically and experimentally for discovery of immune functions. However, little effort has been dedicated to determining which specific system(s) within defense islands limit lytic phage reproduction in clinical bacterial strains. Here, we employed the CRISPR-based Cascade-Cas3 system to delete defense islands in a Pseudomonas aeruginosa clinical isolate to identify mechanisms of lytic phage antagonism. Deletion of one island in a cystic fibrosis-derived clinical isolate sensitized the strain to phages from the Pbunavirus family, which are commonly used as therapeutics. The causal defense system is a Type IIS restriction endonuclease-like protein (ENDPaCF1), common in Pseudomonads, however it lacks an associated methyltransferase typical Type IIS R-M systems. ENDPaCF1 protects bacteria against phages with hypermodified DNA and is surprisingly agnostic to the specific structure of the modification, which is unlike typical type IV restriction endonucleases. In ENDPaCF1, the endonuclease domain is fused to a catalytically inactive Endonuclease III (iEndoIII), a domain that recognizes non-canonical bases to repair DNA in prokaryotes and eukaryotes. We therefore propose that nucleases containing an iEndoIII domain (END nucleases) can sense diverse DNA hypermodifications. Our findings reveal modularity of the sensing and cleavage domains, as expected of a modification-dependent endonucleases. We further show that some hypermodified phages, including Pbunavirus family members and Wrowclawvirus family (Pa5oct-like) of jumbo phages, encode END nuclease inhibitors that directly bind to the nuclease, likely via the iEndoIII domain. These inhibitors are necessary for Pbunavirus to plaque on clinical isolates and sufficient to enable other hypermodified phages to plaque in the presence of this defense system.

microbiology↗

bioRxiv2025

The activation of fibroblast growth factor receptor 1 provides therapeutic benefit in a mouse model of Alzheimer's disease

Alzheimers disease (AD) is a neurodegenerative disease that is characterized by the accumulation of amyloid plaque and neurofibrillary tangles, ultimately impairing multiple cognitive domains. Both plaques and tangles cause neuronal damage and stimulate inflammatory responses in glial cells. The fibroblast growth factor receptor 1 (FGFR1)-mediated signaling pathways support the function of damaged neurons and modulate inflammatory response. The FGFR1 agonists, including Fibroblast growth factor 1 (FGF1) and FG loop peptide (FGL), have been implicated in multiple disease therapy. Whether FGFR1 agonists can improve pathology and cognitive function in AD remains unknown. Here, we showed that administration of FGF1 and FGL to the AD mouse model reversed spatial memory impairment, enhanced neurogenesis, suppressed reactive astrogliosis, and restricted dystrophic neurite. However, only FGF1 treatment reduced the deposition of senile plaque. In microglial culture studies, FGF1 improves the phagocytosis ability of microglia, which contributes to the clearance of plaques. Together, our findings suggested that FGFR1 agonists alleviate pathology and cognitive impairment via immunomodulatory and improve neuronal health in the AD mouse model. HighlightsFGFR1 agonists alleviate memory deficits in AD mice. FGFR1 agonists increase adult neurogenesis and limit dystrophic neurites in AD mice. FGFR1 agonists increase plaque-associated microglia in vivo and promote phagocytosis in vitro.

neuroscience↗

bioRxiv2024

The discovery of 5mC-selective deaminases and their application to ultra-sensitive direct sequencing of methylated sites at base resolution.

Mining phages for new enzymatic activities continues to be important for the development of new tools for biotechnology. In this study, we used MetaGPA--a method linking genotype to phenotype in metagenomic data--to identify deoxycytidine deaminases, a protein family highly associated with cytosine modifications in metaviromes. Unexpectedly, a subset of these deaminases exhibited a preference for 5-methylcytosine (5mC) over cytosine (C) in both mononucleotide and single-stranded DNA substrates. In a methylome sequencing workflow, preferential deamination of 5mC by these enzymes enabled direct conversion of methylated cytosine while completely eliminating any background deamination of unmodified cytosine. This direct conversion allows for precise identification of methylated sites at single-base resolution with unmatched sensitivity enabling broad applications for the simultaneous sequencing of genome and methylome.

genomics↗

bioRxiv2024

KlebPhaCol: A community-driven resource for Klebsiella research identified a novel gut phage order associated with the human gut

The growing threat of multidrug-resistant Klebsiella pneumoniae, coupled with its role in gut colonisation, has intensified the search for new treatments, including bacteriophage therapy. Despite increasing documentation of Klebsiella-targeting phages, clinical applications remain limited, with key phage-bacteria interactions still poorly understood. A major obstacle is fragmented access to well-characterised phage-bacteria pairings, restricting the collective advancement of therapeutic and mechanistic insights. To address this gap, we created the Klebsiella Phage Collection (KlebPhaCol), an open-source resource comprising 53 phages and 74 Klebsiella isolates, all fully characterised. These phages span five families - including a novel order, Felixvirales, associated with the human gut - and target 27 sequence types (including ST258, ST11, ST14) and 28 capsular-locus types (including KL1 and KL2), across six Klebsiella species. Freely accessible at www.klebphacol.org, KlebPhaCol invites the scientific community to both use and contribute to this resource, fostering collaborative research and a deeper understanding of Klebsiella-phage interactions beyond therapeutic use.

microbiology↗

bioRxiv2024

The Taiwan Precision Medicine Initiative: A Cohort for Large-Scale Studies

The Taiwan Precision Medicine Initiative (TPMI), a project initiated by the Academia Sinica in collaboration with 16 major medical centers around Taiwan, has recruited 565,390 participants who consented to provide DNA samples for genetic profiling and grant access to their electronic medical records (EMR) for studies to develop precision medicine. Access to the EMR is both retrospective and prospective, allowing researchers to conduct prospective studies over time. Genetic profiling is done with population-optimized SNP arrays for the Han Chinese populations that enable genetic analyses such as genome-wide association, phenome-wide association, and polygenic risk score studies to evaluate common disease risk and pharmacogenetic response. Furthermore, the TPMI participants agree to be contacted for future research opportunities related to their genetic risks and receive personalized genetic risk profiles with health management recommendations. TPMI has established the TPMI Data Access Platform (TDAP), a central database and analysis platform that both safeguards the security of the data and facilitates academic research. The TPMI is the largest non-European cohort that merges genetic profiles with EMR in the world. With a cohort that can be followed over time, it can be utilized to validate genetic risk prediction models, conduct clinical trials to show the efficacy of risk-based health management, and optimize health policies based on genetic risks. In this report, we describe the TPMI study design, the population and genetic characteristics of the TPMI cohort, and the power it provides to conduct crucial studies in developing precision medicine on a population and personal level. As Han Chinese represent almost 20% of the worlds population, the results of TPMI studies will benefit >1.4 billion people around the world and serve as a model for developing population-based precision medicine.

genetics↗

bioRxiv2024

Tunable, self-contained gene dosage control via proteolytic cleavage of CRISPR-Cas systems

The ability to modulate gene expression through modular and universal genetic tools like CRISPR-Cas has greatly advanced gene therapy for therapeutics and basic science. Yet, the inherent stochasticity of delivery methods cause variation in target gene expression at the single-cell level, limiting their applicability in systems that require more precise expression. Thus, we implement a modular incoherent feedforward loop based on proteolytic cleavage of Cas to reduce gene expression variability against the variability of vector delivery. We target a genome-integrated marker and demonstrate dosage control of gene activation and repression, post-delivery tuning, and RNA-based compatibility of the system. To illustrate therapeutic relevance, we target the gene RAI1, the haploinsufficiency and triplosensitivity of which cause two autism-related syndromes. We demonstrate dosage-controlled gene activation for both human and mouse Rai1 via viral delivery to patient-derived cell lines and mouse cortical neurons. Overall, we established a robust dosage control circuit for uniform gene expression, beneficial for basic and translational research.

synthetic biology↗

bioRxiv2023

Optimal Liquid-Based DNA Preservation for DNA Barcoding of Field-Collected Fungal Specimens

Preserving fungal tissue DNA in the field is essential for molecular ecological research, enabling the study of fungal biodiversity and community dynamics. This study systematically compares two liquid-based preservation solutions, RNAlater and DESS, for their effectiveness in maintaining macrofungi DNA integrity during field collection and storage. The research encompasses both controlled experiments and real-world field collections. In the controlled experiments, two fungal species were preserved in RNAlater and DESS at different temperatures and durations. DNA extraction success rates were high, but DNA quality and quantity metrics exhibited variations across samples. However, both preservation solutions demonstrated their viability for preserving fungal DNA, with no significant differences between them. In the field-collected macrofungi experiment, 200 fungal specimens were collected and preserved in RNAlater and DESS. The DNA extraction success rate was 98%, with a few exceptions. The statistical analysis, including paired and independent t-tests, showed no statistically significant differences in DNA quality and quantity between the two preservation methods for the field-collected fungal samples. Overall, this study provides valuable insights into the effectiveness of RNAlater and DESS for preserving macrofungi DNA in field conditions. Researchers can confidently choose between these methods based on their specific needs, without compromising the integrity of the DNA. This research contributes to the advancement of fungal molecular ecology and has broader implications for DNA preservation strategies in ecological and environmental studies.

ecology↗

bioRxiv2023

Smith-Magenis syndrome protein Rai1 regulates body weight homeostasis through hypothalamic Bdnf-producing neurons and TrkB signalling

Retinoic acid-induced 1 (RAI1) haploinsufficiency causes Smith-Magenis syndrome (SMS), a genetic disorder with symptoms including hyperphagia, hyperlipidemia, severe obesity, and autism phenotypes. RAI1 is a transcriptional regulator with a pan-neural expression pattern and hundreds of downstream targets. The mechanisms linking neural Rai1 to body weight regulation remain unclear. Here we find that hypothalamic brain-derived neurotrophic factor (BDNF) and its downstream signalling are disrupted in SMS (Rai1+/-) mice. Selective Rai1 loss from all BDNF-producing cells or from BDNF-producing neurons in the paraventricular nucleus of the hypothalamus (PVH) induced obesity in mice. Electrophysiological recordings revealed that Rai1 ablation decreased the intrinsic excitability of PVHBDNF neurons. Chronic treatment of SMS mice with LM22A-4 engages neurotrophin downstream signalling and delayed obesity onset. This treatment also partially rescued disrupted lipid profiles, insulin intolerance, and stereotypical repetitive behaviour in SMS mice. These data argue that RAI1 regulates body weight and metabolic function through hypothalamic BDNF-producing neurons and that targeting neurotrophin downstream signalling might improve associated SMS phenotypes.

neuroscience↗

bioRxiv2023

Four additional natural 7-deazaguanine derivatives in phages and how to make them

Bacteriophages and bacteria are engaged in a constant arms race, continually evolving new molecular tools to survive one another. To protect their genomic DNA from restriction enzymes, the most common bacterial defence systems, double-stranded DNA phages have evolved complex modifications that affect all four bases. This study focuses on modifications at position 7 of guanines. Eight derivatives of 7-deazaguanines were identified, including four previously unknown ones: 2-deoxy-7-(methylamino)methyl-7-deazaguanine (mdPreQ1), 2-deoxy-7-(formylamino)methyl-7-deazaguanine (fdPreQ1), 2-deoxy-7-deazaguanine (dDG), and 2-deoxy-7-carboxy-7-deazaguanine (dCDG). These modifications are inserted in DNA by a guanine transglycosylase named DpdA. Three subfamilies of DpdA had been previously characterized: bDpdA, DpdA1, and DpdA2. Two additional subfamilies were identified in this work: DpdA3, which allows for complete replacement of the guanines, and DpdA4, which is specific to archaeal viruses. Transglycosylases have now been identified in all phages and viruses carrying 7-deazaguanine modifications, indicating that the insertion of these modifications is a post-replication event. Three enzymes were predicted to be involved in the biosynthesis of these newly identified DNA modifications: 7-carboxy-7-deazaguanine decarboxylase (DpdL), dPreQ1 formyltransferase (DpdN), and dPreQ1 methyltransferase (DpdM), which was experimentally validated and harbors a unique fold not previously observed for nucleic acid methylases.

genomics↗

bioRxiv2023

Modification of DNA by a viral enzyme and charged tRNA

Bacteriophage enzymes synthesize varied and complex DNA hypermodifications. The enzyme encoded by the phage Mu gene mom is necessary for post-replicative carbamoylmethyl addition to the exocyclic amine of deoxyadenosine in DNA during the lytic phase of the viral life-cycle. The molecular details of this modification reaction, including the molecular origins of the modification itself, have long eluded understanding. Here, we demonstrate that Mom co-opts the translational machinery of the host by harvesting activated glycine from charged tRNAGly to hypermodify adenine. Based on this insight, we report the first in vitro reconstitution of the Mu hypermodification from purified components. Using isotope labeling, we demonstrate that the carbamoyl nitrogen of the Mom modification is derived from the N6 of adenine, indicating an on-base rearrangement of the N6 aminoacylation product, possibly via a cyclic intermediate. Informed by the X-ray crystal structure of Mom, we have probed the location of the active site, identified a novel insertion, and established substrate specificities of the Mom enzyme.

biochemistry↗