Journal of Biological Chemistry最新文献

Aggrecan immobilizes to perineuronal nets through hyaluronan-dependent and independent binding activities.

IF 4.8 2区生物学

Journal of Biological Chemistry Pub Date : 2025-04-22 DOI: 10.1016/j.jbc.2025.108525

Matthew Y Otsuka,Leslie B Essel,Ashis Sinha,Gabrielle Nickerson,Seth M Mejia,Ashley Edge,Russell T Matthews,Samuel Bouyain

{"title":"Aggrecan immobilizes to perineuronal nets through hyaluronan-dependent and independent binding activities.","authors":"Matthew Y Otsuka,Leslie B Essel,Ashis Sinha,Gabrielle Nickerson,Seth M Mejia,Ashley Edge,Russell T Matthews,Samuel Bouyain","doi":"10.1016/j.jbc.2025.108525","DOIUrl":"https://doi.org/10.1016/j.jbc.2025.108525","url":null,"abstract":"Aggrecan (ACAN) is a large, secreted chondroitin sulfate proteoglycan that includes three globular regions named G1, G2, G3, and is decorated with multiple glycosaminoglycan attachments between its G2 and G3 domains. The N-terminal G1 region interacts with the glycosaminoglycan hyaluronan (HA), which is an essential component of the vertebrate extracellular matrix. In the central nervous system, ACAN is found in perineuronal nets (PNNs), honeycomb-like structures that localize to the surface of parvalbumin-positive neurons in specific neural circuits. PNNs regulate the plasticity of the central nervous system, and it is believed that association between ACAN and HA is a foundational event in the assembly of these reticular structures. Here, we report the co-crystal structure of the G1 region of ACAN in the absence and presence of a HA decasaccharide and analyze the importance of the HA-binding activity of ACAN for its integration into PNNs. We demonstrate that the single immunoglobulin domain and the two Link modules that comprise the G1 region form a single structural unit, and that HA is clamped inside a groove that spans the length of the tandem Link domains. Introducing point mutations in the glycosaminoglycan-binding site eliminates HA-binding activity in ACAN, but, surprisingly, only decreases the integration of ACAN into PNNs. Thus, these results suggest that ACAN can be recruited into PNNs independently of its HA-binding activity.","PeriodicalId":15140,"journal":{"name":"Journal of Biological Chemistry","volume":"33 1","pages":"108525"},"PeriodicalIF":4.8,"publicationDate":"2025-04-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143876478","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

The ubiquitin ligase NKLAM promotes apoptosis and suppression of cell growth.

IF 4.8 2区生物学

Journal of Biological Chemistry Pub Date : 2025-04-22 DOI: 10.1016/j.jbc.2025.108527

Paul A Willard,Jacki Kornbluth

{"title":"The ubiquitin ligase NKLAM promotes apoptosis and suppression of cell growth.","authors":"Paul A Willard,Jacki Kornbluth","doi":"10.1016/j.jbc.2025.108527","DOIUrl":"https://doi.org/10.1016/j.jbc.2025.108527","url":null,"abstract":"Natural Killer Lytic Associated Molecule (NKLAM), also known as RNF19b, is a member of the RING-in between-RING-RING (RBR) E3 ubiquitin ligase family and plays a pivotal role in immune regulation. We identified a critical cysteine residue at position 301 essential for NKLAM's ubiquitin ligase function. Site-directed mutagenesis of this residue to serine or alanine abrogated the ligase activity of NKLAM. Utilizing inducible expression systems in two different cell lines, HEK293 embryonic kidney cells and K562 myeloid leukemia cells, we demonstrated that wild-type (WT) NKLAM, but not the catalytically inactive NKLAM alanine mutant (C301A), inhibited cellular proliferation, as evidenced by reduced cell numbers and decreased metabolic activity. Moreover, NKLAM expression led to a significant decrease in the abundance and stability of the proto-oncogene c-Myc, a key regulator of proliferation. NKLAM facilitated the proteasomal degradation of c-Myc, with a reduction in c-Myc half-life from 27 minutes to 12 minutes and restoration of c-Myc levels upon proteasome inhibition. Notably, prolonged NKLAM expression induced apoptosis, measured by annexin-V staining and caspase activation. Strikingly, the serine mutant, C301S, while lacking ubiquitin ligase activity, induced apoptosis comparable to WT NKLAM, highlighting an alternative pathway for NKLAM-mediated inhibition of cellular homeostasis. Our findings indicate that NKLAM is a cytolytic protein with multifaceted roles in cellular proliferation and apoptosis.","PeriodicalId":15140,"journal":{"name":"Journal of Biological Chemistry","volume":"76 1","pages":"108527"},"PeriodicalIF":4.8,"publicationDate":"2025-04-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143876483","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Phosphorylation modulates secondary structure of intrinsically disorder regions in RNA polymerase II.

IF 4.8 2区生物学

Journal of Biological Chemistry Pub Date : 2025-04-22 DOI: 10.1016/j.jbc.2025.108533

Wei Chen,Tatiana N Laremore,Neela H Yennawar,Scott A Showalter

{"title":"Phosphorylation modulates secondary structure of intrinsically disorder regions in RNA polymerase II.","authors":"Wei Chen,Tatiana N Laremore,Neela H Yennawar,Scott A Showalter","doi":"10.1016/j.jbc.2025.108533","DOIUrl":"https://doi.org/10.1016/j.jbc.2025.108533","url":null,"abstract":"The intrinsically disordered C-terminal domain (CTD) of RNA polymerase II contains tandem repeats with the consensus sequence YSPTSPS and coordinates transcription and co-transcriptional events through dynamic phosphorylation patterns. While it has been long hypothesized that phosphorylation induces structural changes in the CTD, a direct comparison of how different phosphorylation patterns modulate the CTD conformation has been limited. Here, we generated two distinct phosphorylation patterns in an essential Drosophila CTD region with the kinase Dyrk1a: one where Ser2 residues are primarily phosphorylated, mimicking the state near transcription termination, and a hyperphosphorylation state where most Ser2, Ser5, and Thr residues are phosphorylated, expanding on our work on Ser5 phosphorylation, which mimics early transcription elongation. Using 13C Direct-Detect NMR, we show that the CTD tends to form transient beta strands and beta turns, which are altered differently by Ser2 and Ser5 phosphorylation. Small angle x-ray scattering (SAXS) revealed no significant changes in the CTD global dimensions even at high phosphorylation levels, contradicting the common assumption of phosphorylation-induced chain expansion. Our findings support a transient beta model in which unphosphorylated CTD adopts transient beta strands at Ser2 during transcription pre-initiation. These transient structures are disrupted by Ser5 phosphorylation in early elongation, and later restored by Ser2 phosphorylation near termination for recruiting beta turn-recognizing termination factors.","PeriodicalId":15140,"journal":{"name":"Journal of Biological Chemistry","volume":"53 1","pages":"108533"},"PeriodicalIF":4.8,"publicationDate":"2025-04-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143876479","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

A high throughput compatible workflow for the biochemical identification and characterisation of molecular glues.

IF 4.8 2区生物学

Journal of Biological Chemistry Pub Date : 2025-04-22 DOI: 10.1016/j.jbc.2025.108526

Ryan Guilbert,Maxime Couturier,Yuanyuan Si,Daniel O' Donovan,David Longmire,Hazel Mak,Paul Clarkson,Argyrides Argyrou

引用次数: 0

Challenging activity and signaling bias in tachykinin NK1 and NK2 receptors by truncated neuropeptides.

IF 4.8 2区生物学

Journal of Biological Chemistry Pub Date : 2025-04-18 DOI: 10.1016/j.jbc.2025.108522

Jacob E Petersen,Artem Pavlovskyi,Jesper J Madsen,Thue W Schwartz,Thomas M Frimurer,Ole H Olsen

{"title":"Challenging activity and signaling bias in tachykinin NK1 and NK2 receptors by truncated neuropeptides.","authors":"Jacob E Petersen,Artem Pavlovskyi,Jesper J Madsen,Thue W Schwartz,Thomas M Frimurer,Ole H Olsen","doi":"10.1016/j.jbc.2025.108522","DOIUrl":"https://doi.org/10.1016/j.jbc.2025.108522","url":null,"abstract":"The tachykinin receptors neurokinin 1 (NK1R) and neurokinin 2 (NK2R) are G protein-coupled receptors that bind preferentially to the natural peptide ligands substance P (SP) and neurokinin A (NKA), respectively. The peptide ligands share a common C-terminal sequence, Phe-X-Gly-Leu-Met-NH2, which contributes to their partial cross-reactivity with each other's non-native receptors. This study examines the impact of truncated tachykinin SP and NKA analogs on signaling activity. SP and NKA were progressively truncated, yielding the shortest versions SP(6-11) and NKA(5-10) with free and acetylated N-terminal. A total of 12 SP and 10 NKA analogs were evaluated for activity in BRET-based cAMP and IP3 accumulation assays targeting both NK1R and NK2R, corresponding to Gs protein and Gq protein activation, respectively. As previously demonstrated, the first three amino acids are dispensable. When activated by SP analogs, NK1R favors activation of Gs over Gq, though this difference diminishes with shorter analogs. In contrast, when NK1R is activated by NKA analogs, the Gq potency exceeds Gs potency by nearly an order of magnitude. For NK2R activation by NKA analogs, there are only minor differences between Gq and Gs potencies, with a slight preference for higher Gq potency. The N-terminal charge status plays a key role, leading to significant differences in analog potency. These findings provide valuable insight into how specific receptor-ligand interactions influence downstream G-protein signaling in GPCRs, which are highly relevant for therapeutic applications. Finally, the proposed \"message-address\" model of neuropeptide signaling is assessed for NK1R and NK2R using truncated SP and NKA analogs.","PeriodicalId":15140,"journal":{"name":"Journal of Biological Chemistry","volume":"34 1","pages":"108522"},"PeriodicalIF":4.8,"publicationDate":"2025-04-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143857340","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Identification and design principles of far-red-absorbing chlorophyll in the light-harvesting complex.

IF 4.8 2区生物学

Journal of Biological Chemistry Pub Date : 2025-04-18 DOI: 10.1016/j.jbc.2025.108518

Keisuke Saito,Makiko Kosugi,Linhao Qiu,Jun Minagawa,Hiroshi Ishikita

{"title":"Identification and design principles of far-red-absorbing chlorophyll in the light-harvesting complex.","authors":"Keisuke Saito,Makiko Kosugi,Linhao Qiu,Jun Minagawa,Hiroshi Ishikita","doi":"10.1016/j.jbc.2025.108518","DOIUrl":"https://doi.org/10.1016/j.jbc.2025.108518","url":null,"abstract":"Photosystem II (PSII) from Prasiola crispa employs a unique ring-shape undecameric light-harvesting complex (Pc-frLHC) in addition to the commonly observed minor monomeric and major trimeric LHCIIs. Each monomer of Pc-frLHC contains four transmembrane helices. In contrast to the typical three-helix LHCIIs that constitute for the peripheral light-harvesting antennas for PSII, Pc-frLHC carries chlorophylls capable of far-red absorption. Combining spectroscopic analyses with a quantum mechanical/molecular mechanical approach, we identified the far-red absorbing chlorophyll(s) in Pc-frLHC, as well as its counterpart in another Trebouxiophyceae alga Coccomyxa sp. Obi (Co-frLHC). Spectroscopic analysis reveals that both complexes exhibit far-red-shifted absorption of chlorophylls at ∼710 nm. In the Pc-frLHC structure, the Chla 603-609 dimer exhibits the strongest excitonic coupling among all apparent chlorophyll dimers. This dimer also exhibits the largest excitation-induced permanent dipole moment along the axis connecting the two chlorophylls, reflecting the most pronounced charge-transfer character. Furthermore, Chla 609 forms the second strongest excitonically coupled dimer with Chla 708, further extending the absorption into the far-red region. The conserved spatial arrangement and orientation of the chlorophyll trimer in Co-frLHC suggest that the Chla 603-609-708 trimer, located in the same frLHC monomer unit, which is predominantly characterized by the Chla 603-609 dimer, provides the structural basis for the far-red absorption in frLHCs.","PeriodicalId":15140,"journal":{"name":"Journal of Biological Chemistry","volume":"7 1","pages":"108518"},"PeriodicalIF":4.8,"publicationDate":"2025-04-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143857339","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Potent activity of prostaglandin J2 on prostanoid DP receptors.

IF 4.8 2区生物学

Journal of Biological Chemistry Pub Date : 2025-04-18 DOI: 10.1016/j.jbc.2025.108523

Kanaho Senoo,Keijo Fukushima,Hitomi Yamamoto,Ayaka Hamaguchi,Akiko Suganami,Harumi Takano,Mayu Yamashita,John W Regan,Yutaka Tamura,Hiromichi Fujino

{"title":"Potent activity of prostaglandin J2 on prostanoid DP receptors.","authors":"Kanaho Senoo,Keijo Fukushima,Hitomi Yamamoto,Ayaka Hamaguchi,Akiko Suganami,Harumi Takano,Mayu Yamashita,John W Regan,Yutaka Tamura,Hiromichi Fujino","doi":"10.1016/j.jbc.2025.108523","DOIUrl":"https://doi.org/10.1016/j.jbc.2025.108523","url":null,"abstract":"Prostaglandin D2 (PGD2), an anti-inflammatory mediator, is acting through Gs-protein coupled D-type prostanoid (DP) receptors. DP receptors are not extensively distributed; in tissues, they are the least abundant among members of the prostanoid receptor family, whereas their primary ligand PGD2 is the main prostanoid in most tissues. PGD2 is dehydrated or isomerized to a number of metabolites enzymatically or non-enzymatically. To understand why many metabolites of PGD2 are produced via different pathways, regular cell-based experiments, Black/Leff operational model calculations, and in silico simulations were utilized. Here we show, among the 5 metabolites of PGD2, prostaglandin J2 (PGJ2) was the most potent metabolite for DP receptors, particularly in the cAMP signaling pathway. This result was attributed to PGJ2 forming an extra, and/or stronger hydrogen bond by more negatively charged carbonyl in the cyclopentene ring with DP receptors than PGD2. Therefore, when PGD2 is released into the blood, it would activate DP receptors, which are then continuously activated by PGJ2 to sustain the DP receptor/cAMP-mediated signaling pathway. Thus, the anti-inflammatory effects of PGD2 may be taken over/out competed and/or even enhanced by PGJ2. Here, PGJ2 was found to be a standout mediator of cAMP-mediated signaling pathway, that induces more potent and prolonged DP receptor-activities as a biased ligand, possibly for resolving the inflammatory reaction. Moreover, since each metabolite showed different property, these results provide insight into why many metabolites of PGD2 are produced, and the miscellaneous physiological roles induced by the main prostanoid in most tissues through the least abundant DP receptors.","PeriodicalId":15140,"journal":{"name":"Journal of Biological Chemistry","volume":"64 1","pages":"108523"},"PeriodicalIF":4.8,"publicationDate":"2025-04-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143857469","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Optimization of the PROTAC linker region of the proteasome substrate receptor hRpn13 rationalized structural modeling with molecular dynamics.

IF 4.8 2区生物学

Journal of Biological Chemistry Pub Date : 2025-04-18 DOI: 10.1016/j.jbc.2025.108520

Xiuxiu Lu,Venkata R Sabbasani,Bakar Hassan,Rolf E Swenson,Kylie J Walters

{"title":"Optimization of the PROTAC linker region of the proteasome substrate receptor hRpn13 rationalized structural modeling with molecular dynamics.","authors":"Xiuxiu Lu,Venkata R Sabbasani,Bakar Hassan,Rolf E Swenson,Kylie J Walters","doi":"10.1016/j.jbc.2025.108520","DOIUrl":"https://doi.org/10.1016/j.jbc.2025.108520","url":null,"abstract":"Proteasome substrate receptor hRpn13 is a promising target for cancer therapy. hRpn13 PROTACs induce apoptosis by targeting the hRpn13 proteolytic product hRpn13Pru, which contains an intact ubiquitin- and proteasome-binding Pru domain. We generated a PROTAC series based on hRpn13Pru-targeting XL5 by varying the linker that connects it to a warhead against the VHL-based ubiquitin E3 ligase machinery. Among eight tested derivatives, XL5-VHL-7 with a -(CH2)5- alkyl linker promoted hRpn13Pru degradation and induced cellular apoptosis with 2-fold improved potency compared to the original PROTAC. By using this PROTAC series with slight chemical modifications in the linker region, we were able to evaluate the efficacy of structural modeling with molecular dynamics for refining PROTACs. Overall, we found that the experimental data correlated with efficacy predictions based on molecular dynamics and structural modeling. Moreover, we could observe hRpn13:PROTAC:VHL complexes by 2D NMR that support the structural modeling and stronger affinity of XL5-VHL-7 compared to the original hRpn13 PROTAC. Our NMR data further indicate that hRpn13 Pru affinity for XL5-VHL-7 is higher within the VHL complex present than with XL5-VHL-7 alone. Altogether, we develop an hRpn13 PROTAC with 2-fold increased potency by optimizing the linker and demonstrate the current benefit and limitations for including modeling with molecular dynamics to aid PROTAC optimization.","PeriodicalId":15140,"journal":{"name":"Journal of Biological Chemistry","volume":"43 1","pages":"108520"},"PeriodicalIF":4.8,"publicationDate":"2025-04-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143857341","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

MitoSNO inhibits mitochondrial hydrogen peroxide (mtH2O2) generation by α-ketoglutarate dehydrogenase (KGDH).

IF 4.8 2区生物学

Journal of Biological Chemistry Pub Date : 2025-04-16 DOI: 10.1016/j.jbc.2025.108510

Olivia Chalifoux,Samantha Sterman,Ben Faerman,Meijing Li,Stephanie Trezza,Marek Michalak,Luis B Agellon,Ryan J Mailloux

{"title":"MitoSNO inhibits mitochondrial hydrogen peroxide (mtH2O2) generation by α-ketoglutarate dehydrogenase (KGDH).","authors":"Olivia Chalifoux,Samantha Sterman,Ben Faerman,Meijing Li,Stephanie Trezza,Marek Michalak,Luis B Agellon,Ryan J Mailloux","doi":"10.1016/j.jbc.2025.108510","DOIUrl":"https://doi.org/10.1016/j.jbc.2025.108510","url":null,"abstract":"Here, we demonstrate mitochondrial hydrogen peroxide (mtH2O2) production by α-ketoglutarate dehydrogenase (KGDH) can be inhibited by MitoSNO, alleviating lipotoxicity. MitoSNO in the nanomolar range inhibits mtH2O2 by ∼50% in isolated liver mitochondria without disrupting respiration, whereas the mitochondria-selective derivative used to synthesize MitoSNO, mitochondria-selective N-acetyl-penicillamine (MitoNAP), had no effect on either mtH2O2 generation or oxidative phosphorylation (OxPhos). Additionally, mtH2O2 generation in isolated liver mitochondria was almost abolished when MitoSNO was administered in the low micromolar range. The potent inhibitory effect of MitoSNO was comparable to 2-keto-3-methyl-valeric acid (KMV) and valproic acid (VA), selective inhibitors for KGDH-mediate mH2O2 production. S1QEL 1.1 (S1) and S3QEL (S3), which are known to selectively suppress mtH2O2 genesis through inhibition of complex I and complex III respectively, without disrupting respiration, had little to no effect on mtH2O2 production by liver mitochondria. We also identified it was a major mtH2O2 source as well but MitoSNO and MitoNAP did not affect mtH2O2 production by this ETC-linked enzyme. The MitoSNO also suppressed mtH2O2 production and partially rescued mitochondrial respiration in Huh-7 cells subjected to palmitate (PA) and fructose (Fruc) induced lipotoxicity. MitoSNO also prevented cell death and abrogated intrahepatic lipid accumulation in these Huh-7 cells. MitoSNO nullified mtH2O2 overgeneration and partially rescued OxPhos in liver mitochondria from mice fed a high fat diet (HFD). Our findings demonstrate that MitoSNO interferes with mtH2O2 production through KGDH S-nitrosation and may be useful in alleviating non-alcoholic fatty liver disease (NAFLD).","PeriodicalId":15140,"journal":{"name":"Journal of Biological Chemistry","volume":"45 1","pages":"108510"},"PeriodicalIF":4.8,"publicationDate":"2025-04-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143851014","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

High frequency transcription leads to rapid R-loop formation.

IF 4.8 2区生物学

Journal of Biological Chemistry Pub Date : 2025-04-16 DOI: 10.1016/j.jbc.2025.108514

Bradleigh Palmer,Chun-Ying Lee,Leya Yang,Tapas Paul,Sua Myong

{"title":"High frequency transcription leads to rapid R-loop formation.","authors":"Bradleigh Palmer,Chun-Ying Lee,Leya Yang,Tapas Paul,Sua Myong","doi":"10.1016/j.jbc.2025.108514","DOIUrl":"https://doi.org/10.1016/j.jbc.2025.108514","url":null,"abstract":"R-loops are transcriptionally generated three-stranded nucleic acid structures where the mRNA hybridizes with template DNA, leaving a displaced single-stranded non-template DNA loop. Previously, we demonstrated that R-loop and subsequent G-quadruplex formation upregulate transcription. However, the mechanistic basis of how transcription activity generates R-loop formation is unknown. Here, we investigate the kinetics of transcription and its impact on R-loop formation using single-molecule fluorescence resonance energy transfer (smFRET) and electrophoretic mobility shift assay (EMSA). We show that R-loop formation is tuned by the frequency and the rate of transcription, controlled by the RNAP and NTP concentrations, respectively. We provide a plausible mechanism in which gradually increasing the duration of the promoter opening leads to the R-loop formation. Through stochastic simulation, we demonstrate that the frequency of transcription primarily governs R-loop formation. This work highlights the intricate balance between transcription dynamics and R-loop formation, providing new insights into the structure-function relationship.","PeriodicalId":15140,"journal":{"name":"Journal of Biological Chemistry","volume":"108 1","pages":"108514"},"PeriodicalIF":4.8,"publicationDate":"2025-04-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143851029","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0