Journal of Biological Chemistry最新文献

{"title":"Microcurrent stimulation induces cell death in p53-mutant and 5-FU-resistant breast cancer.","authors":"Tomohito Tanihara, Yuya Yoshida, Takashi Ogino, Yuma Terada, Fumiaki Tsurusaki, Keika Hamasaki, Kaita Otsuki, Kohei Fukuoka, Kosuke Oyama, Akito Tsuruta, Kengo Hamamura, Kouta Mayanagi, Satoru Koyanagi, Yuichi Murakami, Mayumi Ono, Michihiko Kuwano, Shigehiro Ohdo, Naoya Matsunaga","doi":"10.1016/j.jbc.2025.110414","DOIUrl":"https://doi.org/10.1016/j.jbc.2025.110414","url":null,"abstract":"<p><p>5-Fluorouracil (5-FU) is a commonly used chemotherapeutic agent for breast cancer. Its efficacy relies on the function of p53, and mutations in p53 contribute to the development of resistance during 5-FU chemotherapy. Here, we report that microcurrent stimulation (MCS) of a p53-mutant breast cancer cell line induces p53-mediated cell death. Although MDA-MB-231 and MDA-MB-468 cells, both human breast cancer cell lines, are less sensitive to 5-FU due to p53 mutations, MCS (300 μA for 30 min) induced apoptosis in these cells and improved the antitumor effect of 5-FU in tumor-bearing mice. MCS-induced apoptosis was mediated by an increase in intracellular Cu²⁺ ions and reactive oxygen species, along with the concurrent transcriptional enhancement of pro-apoptotic genes by p53. Furthermore, MCS induced apoptosis in MDA-MB-231 cells that had developed resistance to 5-FU and inhibited tumor growth in tumor-bearing mice with reduced 5-FU sensitivity. These findings suggest that an approach involving MCS could serve as a foundation for developing breast cancer treatment strategies to overcome p53 mutations.</p>","PeriodicalId":15140,"journal":{"name":"Journal of Biological Chemistry","volume":" ","pages":"110414"},"PeriodicalIF":4.0,"publicationDate":"2025-06-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144505806","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}

{"title":"A molecular basis underpinning TRBV28+ T cell receptor recognition of MR1-antigen.","authors":"Wael Awad,Nicholas A Gherardin,Lisa Ciacchi,Andrew N Keller,Ligong Liu,David P Fairlie,James McCluskey,Dale I Godfrey,Jamie Rossjohn","doi":"10.1016/j.jbc.2025.110416","DOIUrl":"https://doi.org/10.1016/j.jbc.2025.110416","url":null,"abstract":"Mucosal-associated invariant T (MAIT) cells express a TRAV1-2+ T cell receptor (TCR) that recognises microbial vitamin B2-derivatives presented by the MHC class I-related molecule, MR1. Most MAIT TCRs incorporate a biased TCR-β repertoire, predominantly TRBV20-1 and TRBV6, but some utilise other TRBV genes, including TRBV28. A second conserved, albeit less frequent TRAV36+ TRBV28+ T cell population exhibits MAIT-like phenotypic features but use a markedly distinct mode of MR1-antigen-recognition compared to MAIT TCR-MR1 binding. Nevertheless, our understanding of how differing TCR gene usage results in altered MR1 binding modes remains incomplete. Here, binding studies demonstrated differential affinities and antigen-specificities between TRBV6+ and TRBV28+ MR1-restricted TCRs. Alanine-scanning mutagenesis on the TRAV36-TRBV28 TCR, revealed a strong dependence on germline-encoded residues within the highly selected CDR3α loop, similar to TRAV1-2- TRBV6 TCRs, and further alanine-scanning mutagenesis experiments demonstrate differential energetic footprints by these TCRs atop MR1. We determined the crystal structure of a MAIT TRAV1-2-TRBV28+ TCR-MR1-5-OP-RU ternary complex. This structure revealed a docking mode conserved amongst other TRAV1-2+ MAIT TCRs, with the TRBV28-encoded TCR-β chain adopting highly distinct docking modes between the TRAV1-2+ and TRAV36+ TCRs. This indicates that the TCR-α chain dictates the positioning and role of the TCR-β chain. Taken together, these findings provide new molecular insights into MR1-Ag driven selection of paired TCR-α and TCR-β chains.","PeriodicalId":15140,"journal":{"name":"Journal of Biological Chemistry","volume":"36 1","pages":"110416"},"PeriodicalIF":4.8,"publicationDate":"2025-06-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144504549","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}

{"title":"Microcurrent stimulation induces cell death in p53-mutant and 5-FU-resistant breast cancer.","authors":"Tomohito Tanihara,Yuya Yoshida,Takashi Ogino,Yuma Terada,Fumiaki Tsurusaki,Keika Hamasaki,Kaita Otsuki,Kohei Fukuoka,Kosuke Oyama,Akito Tsuruta,Kengo Hamamura,Kouta Mayanagi,Satoru Koyanagi,Yuichi Murakami,Mayumi Ono,Michihiko Kuwano,Shigehiro Ohdo,Naoya Matsunaga","doi":"10.1016/j.jbc.2025.110414","DOIUrl":"https://doi.org/10.1016/j.jbc.2025.110414","url":null,"abstract":"5-Fluorouracil (5-FU) is a commonly used chemotherapeutic agent for breast cancer. Its efficacy relies on the function of p53, and mutations in p53 contribute to the development of resistance during 5-FU chemotherapy. Here, we report that microcurrent stimulation (MCS) of a p53-mutant breast cancer cell line induces p53-mediated cell death. Although MDA-MB-231 and MDA-MB-468 cells, both human breast cancer cell lines, are less sensitive to 5-FU due to p53 mutations, MCS (300 μA for 30 min) induced apoptosis in these cells and improved the antitumor effect of 5-FU in tumor-bearing mice. MCS-induced apoptosis was mediated by an increase in intracellular Cu2+ ions and reactive oxygen species, along with the concurrent transcriptional enhancement of pro-apoptotic genes by p53. Furthermore, MCS induced apoptosis in MDA-MB-231 cells that had developed resistance to 5-FU and inhibited tumor growth in tumor-bearing mice with reduced 5-FU sensitivity. These findings suggest that an approach involving MCS could serve as a foundation for developing breast cancer treatment strategies to overcome p53 mutations.","PeriodicalId":15140,"journal":{"name":"Journal of Biological Chemistry","volume":"18 1","pages":"110414"},"PeriodicalIF":4.8,"publicationDate":"2025-06-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144504552","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}

{"title":"Molecular Choreography of E1 Enzymes in Ubiquitin-like Protein Cascades: New Insights into Dynamics and Specificity.","authors":"Caleb M Stratton,Pirouz Ebadi,Shaun K Olsen","doi":"10.1016/j.jbc.2025.110415","DOIUrl":"https://doi.org/10.1016/j.jbc.2025.110415","url":null,"abstract":"In 2004, Aaron Ciechanover, Avram Hershko, and Irwin Rose were awarded the Nobel Prize in Chemistry for their groundbreaking work uncovering the stepwise, ATP-dependent degradation of cellular proteins. These studies laid the foundation for understanding ubiquitin (Ub) and ubiquitin-like (Ubl) proteins, an evolutionary conserved family of modifiers that mediate diverse cellular processes. The Ub/Ubl system operates through a reaction cascade involving E1 activating, E2 conjugating, and E3 ligating enzymes. As the initiating enzymes, E1s catalyze Ubl adenylation, thiolation, and thioester transfer to their cognate E2s. Despite their conserved architecture, E1s exhibit strict specificity for different Ubls and E2s, a critical feature for maintaining cellular homeostasis. While the molecular mechanisms underlying E1 interactions and activities remain incompletely understood, structural studies have provided key insights into the dynamic changes that accompany Ubl activation and transfer. This review highlights recent structures that build upon foundational biochemical research, elucidating the determinants of activity, specificity, and novel regulatory mechanisms governing E1 enzymes. We examine how conformational changes drive the transition from an adenylate-competent to a thioester-competent state and how these rearrangements facilitate interactions with Ubls and E2s while advancing the reaction cycle. Additionally, we explore recent insights into a prokaryotic E1-E2-like fusion that is structurally homologous to the noncanonical eukaryotic E1 ATG7, revealing its role in activating and conjugating a non-Ubl substrate and its implications for the evolutionarily trajectory of Ubl cascades. Finally, we discuss the current landscape of E1 inhibitors under investigation as potential anti-cancer therapies, as well as prospects for future investigations.","PeriodicalId":15140,"journal":{"name":"Journal of Biological Chemistry","volume":"3 1","pages":"110415"},"PeriodicalIF":4.8,"publicationDate":"2025-06-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144504561","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}

{"title":"Aberrant activation of IL-6/JAK/STAT3/FOSL1 signaling induces renal abnormalities in a Xenopus model of Joubert syndrome-related disorders.","authors":"Udval Uuganbayar, Hiromasa Ninomiya, Issei S Shimada, Chisato Yamada, Mayu Kanie, Shinji Kawai, Takahiro Asai, Toru Miyoshi-Akiyama, Masayuki Itoh, Yutaka Hashimoto, Yoichi Kato","doi":"10.1016/j.jbc.2025.110413","DOIUrl":"https://doi.org/10.1016/j.jbc.2025.110413","url":null,"abstract":"<p><p>CEP290 gene mutations are linked to Joubert syndrome-related disorders (JSRD) which present with various symptoms including brain malformation, retinal degeneration and kidney disorders. It remains unclear how JSRD patients with CEP290 gene mutations lead to kidney disorders, particularly polycystic kidney disease including nephronophthisis (NPH). To address this question, Xenopus CEP290 (xCEP290) was depleted using morpholino oligonucleotides against xCEP290 in Xenopus embryos. xCEP290 morphants exhibited edema and dilated pronephric tubule, indicative of renal dysfunction. Next, RNA-seq analysis was performed to explore which signals and molecules are important for the formation of dilated pronephric tubule observed in the xCEP290 morphant kidney. The hallmark gene set associated with the IL-6/JAK/STAT3 signaling pathway was up-regulated in xCEP290 morphant kidney, and inhibition of this signaling by JAK inhibitor ruxolitinib suppressed the dilated pronephric tubule in xCEP290 morphants. Furthermore, the expression level of transcription factor Xenopus FOSL1 (xFOSL1), whose gene expression is regulated by IL-6 signaling, was up-regulated in xCEP290 morphant kidney, and overexpression of xFOSL1 induced pronephric tubular dilation. These results together revealed that abnormal activation of IL-6/JAK/STAT3/FOSL1 signal axis is responsible for dilated pronephric tubule resembling cystic lesions observed in polycystic kidney disease of JSRD patients with CEP290 gene mutations.</p>","PeriodicalId":15140,"journal":{"name":"Journal of Biological Chemistry","volume":" ","pages":"110413"},"PeriodicalIF":4.0,"publicationDate":"2025-06-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144505804","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}

{"title":"A molecular basis underpinning TRBV28⁺ T cell receptor recognition of MR1-antigen.","authors":"Wael Awad, Nicholas A Gherardin, Lisa Ciacchi, Andrew N Keller, Ligong Liu, David P Fairlie, James McCluskey, Dale I Godfrey, Jamie Rossjohn","doi":"10.1016/j.jbc.2025.110416","DOIUrl":"https://doi.org/10.1016/j.jbc.2025.110416","url":null,"abstract":"<p><p>Mucosal-associated invariant T (MAIT) cells express a TRAV1-2⁺ T cell receptor (TCR) that recognises microbial vitamin B2-derivatives presented by the MHC class I-related molecule, MR1. Most MAIT TCRs incorporate a biased TCR-β repertoire, predominantly TRBV20-1 and TRBV6, but some utilise other TRBV genes, including TRBV28. A second conserved, albeit less frequent TRAV36⁺ TRBV28⁺ T cell population exhibits MAIT-like phenotypic features but use a markedly distinct mode of MR1-antigen-recognition compared to MAIT TCR-MR1 binding. Nevertheless, our understanding of how differing TCR gene usage results in altered MR1 binding modes remains incomplete. Here, binding studies demonstrated differential affinities and antigen-specificities between TRBV6⁺ and TRBV28⁺ MR1-restricted TCRs. Alanine-scanning mutagenesis on the TRAV36-TRBV28 TCR, revealed a strong dependence on germline-encoded residues within the highly selected CDR3α loop, similar to TRAV1-2- TRBV6 TCRs, and further alanine-scanning mutagenesis experiments demonstrate differential energetic footprints by these TCRs atop MR1. We determined the crystal structure of a MAIT TRAV1-2-TRBV28⁺ TCR-MR1-5-OP-RU ternary complex. This structure revealed a docking mode conserved amongst other TRAV1-2⁺ MAIT TCRs, with the TRBV28-encoded TCR-β chain adopting highly distinct docking modes between the TRAV1-2⁺ and TRAV36⁺ TCRs. This indicates that the TCR-α chain dictates the positioning and role of the TCR-β chain. Taken together, these findings provide new molecular insights into MR1-Ag driven selection of paired TCR-α and TCR-β chains.</p>","PeriodicalId":15140,"journal":{"name":"Journal of Biological Chemistry","volume":" ","pages":"110416"},"PeriodicalIF":4.0,"publicationDate":"2025-06-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144505803","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}

{"title":"Spatiotemporal fluctuations in fluorescence intensity of rhodamine phalloidin-labeled actin filaments.","authors":"Kenta Toshino,Yosuke Yamazaki,Shunsuke Ando,Ryuichi Kaneda,Kazunori Ono,Takahiro Suzuki,Saku T Kijima,Taro Q P Uyeda","doi":"10.1016/j.jbc.2025.110417","DOIUrl":"https://doi.org/10.1016/j.jbc.2025.110417","url":null,"abstract":"Phalloidin is widely used for fluorescent labeling of actin filaments. We observed ADP-actin filaments labeled with rhodamine-phalloidin or Alexa488-phalloidin in vitro and discovered that the fluorescence intensities along the filaments showed a mottled pattern of bright and dark regions. Filaments labeled with sub-stoichiometric rhodamine-phalloidin exhibited more significant fluorescence inhomogeneities than those labeled with excess rhodamine-phalloidin. Because the quantum yield of Alexa488 fluorescence is hardly affected by the environment, we concluded that the inhomogeneities arise from non-uniform phalloidin binding density rather than locally inhomogeneous quantum yield of the fluorophores. Simulations assuming random rhodamine-phalloidin binding alone partially produced fluorescence inhomogeneities, but the degree of inhomogeneities was significantly smaller than the experimental results. Furthermore, filaments co-labeled with rhodamine-phalloidin and Alexa488-phalloidin showed a positive correlation in fluorescence intensities of rhodamine and Alexa488. Moreover, addition of Pi suppressed the fluorescence inhomogeneities and the correlation between the rhodamine and Alexa488 fluorescence intensities. These results indicated that two mechanisms contribute to the non-uniform binding density of phalloidin: (i) stochastic binding and (ii) local differences in phalloidin binding affinity caused by Pi-sensitive structural polymorphism of actin filaments. This structural polymorphism may also affect the binding of various actin-binding proteins, contributing to the functional differentiation of actin filaments in vivo. Moreover, those mottled fluorescence patterns dynamically fluctuated over time. These temporal fluorescence fluctuations required glucose and glucose oxidase but were suppressed by Trolox, likely reflecting photophysical properties of fluorophores influenced by oxygen scavengers and triplet-state quenchers. Taken together, we provide new insights into the structural polymorphism of actin filaments.","PeriodicalId":15140,"journal":{"name":"Journal of Biological Chemistry","volume":"11 1","pages":"110417"},"PeriodicalIF":4.8,"publicationDate":"2025-06-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144504551","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}

{"title":"Aberrant activation of IL-6/JAK/STAT3/FOSL1 signaling induces renal abnormalities in a Xenopus model of Joubert syndrome-related disorders.","authors":"Udval Uuganbayar,Hiromasa Ninomiya,Issei S Shimada,Chisato Yamada,Mayu Kanie,Shinji Kawai,Takahiro Asai,Toru Miyoshi-Akiyama,Masayuki Itoh,Yutaka Hashimoto,Yoichi Kato","doi":"10.1016/j.jbc.2025.110413","DOIUrl":"https://doi.org/10.1016/j.jbc.2025.110413","url":null,"abstract":"CEP290 gene mutations are linked to Joubert syndrome-related disorders (JSRD) which present with various symptoms including brain malformation, retinal degeneration and kidney disorders. It remains unclear how JSRD patients with CEP290 gene mutations lead to kidney disorders, particularly polycystic kidney disease including nephronophthisis (NPH). To address this question, Xenopus CEP290 (xCEP290) was depleted using morpholino oligonucleotides against xCEP290 in Xenopus embryos. xCEP290 morphants exhibited edema and dilated pronephric tubule, indicative of renal dysfunction. Next, RNA-seq analysis was performed to explore which signals and molecules are important for the formation of dilated pronephric tubule observed in the xCEP290 morphant kidney. The hallmark gene set associated with the IL-6/JAK/STAT3 signaling pathway was up-regulated in xCEP290 morphant kidney, and inhibition of this signaling by JAK inhibitor ruxolitinib suppressed the dilated pronephric tubule in xCEP290 morphants. Furthermore, the expression level of transcription factor Xenopus FOSL1 (xFOSL1), whose gene expression is regulated by IL-6 signaling, was up-regulated in xCEP290 morphant kidney, and overexpression of xFOSL1 induced pronephric tubular dilation. These results together revealed that abnormal activation of IL-6/JAK/STAT3/FOSL1 signal axis is responsible for dilated pronephric tubule resembling cystic lesions observed in polycystic kidney disease of JSRD patients with CEP290 gene mutations.","PeriodicalId":15140,"journal":{"name":"Journal of Biological Chemistry","volume":"23 1","pages":"110413"},"PeriodicalIF":4.8,"publicationDate":"2025-06-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144504553","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}

{"title":"The Poly(A) Polymerase Star-PAP is Regulated by Stably Associated Phosphoinositide Messengers.","authors":"Tianmu Wen,Mo Chen,Vincent L Cryns,Richard A Anderson","doi":"10.1016/j.jbc.2025.110412","DOIUrl":"https://doi.org/10.1016/j.jbc.2025.110412","url":null,"abstract":"Star-PAP is a noncanonical poly(A) polymerase that controls gene expression. Star-PAP was previously reported to bind PIPKI⍺ and its product PI(4,5)P2, which regulate Star-PAP activity and expression of specific genes. Recent studies have revealed a nuclear p53-phosphoinositide signaling pathway in which the phosphatidylinositol (PI) transfer proteins (PITPs) and phosphoinositide kinases/phosphatases bind p53 to sequentially modify p53-linked phosphoinositides and regulate p53 function. Here we demonstrate that multiple phosphoinositides are also coupled to Star-PAP in response to stress. This pathway is initiated by PITP⍺/β binding to Star-PAP, and the Star-PAP-phosphoinositide complexes are sequentially modified by PI4KII⍺, PIPKI⍺, IPMK, and PTEN. The formation of Star-PAP-phosphoinositide complexes enhances the association of the small heat shock proteins HSP27 and ⍺B-crystallin with Star-PAP. Knockdown of the PITPs, PIP kinases, or HSP27 reduces the expression of Star-PAP targets. Our results demonstrate that PITP⍺/β play a key role in the assembly of Star-PAP-phosphoinositide complexes that are sequentially interconverted by PIP kinases/phosphatases and recruit the small heat shock proteins to these complexes to regulate Star-PAP activity in response to stress.","PeriodicalId":15140,"journal":{"name":"Journal of Biological Chemistry","volume":"22 1","pages":"110412"},"PeriodicalIF":4.8,"publicationDate":"2025-06-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144504554","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}

{"title":"Single-subunit RNA polymerases, KpnP, Ro45Iw, and CD23823, with precise terminal synthesis.","authors":"Haruka Takatsuki, Ryota Miyachi, Kaito Seo, Katsumi Hagino, Norikazu Ichihashi","doi":"10.1016/j.jbc.2025.110359","DOIUrl":"10.1016/j.jbc.2025.110359","url":null,"abstract":"<p><p>T7 RNA polymerase (RNAP) is a highly active single-peptide polymerase that is widely used for in vitro RNA synthesis. T7 RNAP synthesizes RNA with a heterogeneous 3'-end, which sometimes causes problems. To date, some RNAPs (e.g., KP34 and Syn5) that synthesize RNA with more precise 3'-ends have been reported. However, they have their own characteristics and thus do not fully substitute T7 RNAP. To increase the number of usable RNAP repertoires, we searched for other RNAPs and their promoters in the phage RNAP database. From the first screening of nine RNAPs, we selected three RNAPs, named KpnP, Ro45Iw, and CD23823, together with their promoter sequences. Characterization of recombinant RNAPs revealed that compared to T7, these three polymerases exhibited similar RNA synthesis activities but preferred a slightly lower temperature. Additionally, CD23823 exhibited a slightly higher salt tolerance. Deep sequencing of the 5'- and 3'-termini of the synthesized RNA revealed that the three RNAPs, particularly CD23823, produced more homogeneous termini than T7 RNAP. These results indicate that these previously uncharacterized RNAPs, especially CD23823, are useful for RNA synthesis that requires precise 5'- and 3'-end sequences.</p>","PeriodicalId":15140,"journal":{"name":"Journal of Biological Chemistry","volume":" ","pages":"110359"},"PeriodicalIF":4.0,"publicationDate":"2025-06-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144496775","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}