Quantitative parameters of productive transcription on T5 N25–based promoters are modulated by the initial transcribed sequence and template supercoiling.
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| Title: | Quantitative parameters of productive transcription on T5 N25–based promoters are modulated by the initial transcribed sequence and template supercoiling. |
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| Authors: | Hsu, Lilian M.1 lhsu@mtholyoke.edu, Han, N. Natalie1 nhan@capitalhealth.org |
| Source: | Journal of Biological Chemistry. Oct2025, Vol. 301 Issue 10, p1-20. 20p. |
| Subjects: | Genetic transcription, RNA synthesis, RNA polymerases, Promoters (Genetics), Genetic transcription regulation |
| Abstract: | Productive initiation on the escape rate–limited T5 phage N25 promoter is subject to substantial modulation by the initial transcribed sequence (ITS). It is further compromised by the formation of two classes of open complexes—productive and unproductive. To decipher their roles, we performed single-cycle transcription assays under RNA polymerase (RNAP)–limiting conditions to quantitatively determine the rate of promoter escape and the productive fraction of RNAP open complexes formed at four N25-ITS variant promoters. Each promoter variant was transcribed from three different template conformations, that is, two fragment templates of different lengths and a supercoiled plasmid DNA template. In addition, each time-course transcription reaction was performed in parallel without or with GreB. Our results indicate that ITS variation greatly impacts both parameters, which together determine the extent of productive RNA synthesis from a promoter. Further, both parameters are highly stimulated by template supercoiling, which yields a higher fraction of productive complexes that undergo promoter escape at a faster rate. In contrast, the effect of GreB is selective, showing little effect on RNAP partitioning but increasing the escape rate of N25 variants bearing non-native ITSs. Analysis of the abortive RNA synthesis kinetics on the highly abortive N25anti(-A) promoter reveals the existence of an unproductive ITC making a 7-nt abortive RNA continuously. Based on our new kinetic data and recently published structural information on promoter complexes, we propose for T5 N25 promoters a mechanism of transcription initiation-promoter escape consistent with the roles of the ITS and template supercoiling. [ABSTRACT FROM AUTHOR] |
| Copyright of Journal of Biological Chemistry is the property of Elsevier B.V. and its content may not be copied or emailed to multiple sites without the copyright holder's express written permission. Additionally, content may not be used with any artificial intelligence tools or machine learning technologies. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.) | |
| Database: | Engineering Source |
| FullText | Text: Availability: 0 |
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| Header | DbId: egs DbLabel: Engineering Source An: 189110844 AccessLevel: 6 PubType: Academic Journal PubTypeId: academicJournal PreciseRelevancyScore: 0 |
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| Items | – Name: Title Label: Title Group: Ti Data: Quantitative parameters of productive transcription on T5 N25–based promoters are modulated by the initial transcribed sequence and template supercoiling. – Name: Author Label: Authors Group: Au Data: <searchLink fieldCode="AR" term="%22Hsu%2C+Lilian+M%2E%22">Hsu, Lilian M.</searchLink><relatesTo>1</relatesTo><i> lhsu@mtholyoke.edu</i><br /><searchLink fieldCode="AR" term="%22Han%2C+N%2E+Natalie%22">Han, N. Natalie</searchLink><relatesTo>1</relatesTo><i> nhan@capitalhealth.org</i> – Name: TitleSource Label: Source Group: Src Data: <searchLink fieldCode="JN" term="%22Journal+of+Biological+Chemistry%22">Journal of Biological Chemistry</searchLink>. Oct2025, Vol. 301 Issue 10, p1-20. 20p. – Name: Subject Label: Subjects Group: Su Data: <searchLink fieldCode="DE" term="%22Genetic+transcription%22">Genetic transcription</searchLink><br /><searchLink fieldCode="DE" term="%22RNA+synthesis%22">RNA synthesis</searchLink><br /><searchLink fieldCode="DE" term="%22RNA+polymerases%22">RNA polymerases</searchLink><br /><searchLink fieldCode="DE" term="%22Promoters+%28Genetics%29%22">Promoters (Genetics)</searchLink><br /><searchLink fieldCode="DE" term="%22Genetic+transcription+regulation%22">Genetic transcription regulation</searchLink> – Name: Abstract Label: Abstract Group: Ab Data: Productive initiation on the escape rate–limited T5 phage N25 promoter is subject to substantial modulation by the initial transcribed sequence (ITS). It is further compromised by the formation of two classes of open complexes—productive and unproductive. To decipher their roles, we performed single-cycle transcription assays under RNA polymerase (RNAP)–limiting conditions to quantitatively determine the rate of promoter escape and the productive fraction of RNAP open complexes formed at four N25-ITS variant promoters. Each promoter variant was transcribed from three different template conformations, that is, two fragment templates of different lengths and a supercoiled plasmid DNA template. In addition, each time-course transcription reaction was performed in parallel without or with GreB. Our results indicate that ITS variation greatly impacts both parameters, which together determine the extent of productive RNA synthesis from a promoter. Further, both parameters are highly stimulated by template supercoiling, which yields a higher fraction of productive complexes that undergo promoter escape at a faster rate. In contrast, the effect of GreB is selective, showing little effect on RNAP partitioning but increasing the escape rate of N25 variants bearing non-native ITSs. Analysis of the abortive RNA synthesis kinetics on the highly abortive N25anti(-A) promoter reveals the existence of an unproductive ITC making a 7-nt abortive RNA continuously. Based on our new kinetic data and recently published structural information on promoter complexes, we propose for T5 N25 promoters a mechanism of transcription initiation-promoter escape consistent with the roles of the ITS and template supercoiling. [ABSTRACT FROM AUTHOR] – Name: AbstractSuppliedCopyright Label: Group: Ab Data: <i>Copyright of Journal of Biological Chemistry is the property of Elsevier B.V. and its content may not be copied or emailed to multiple sites without the copyright holder's express written permission. Additionally, content may not be used with any artificial intelligence tools or machine learning technologies. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract.</i> (Copyright applies to all Abstracts.) |
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| RecordInfo | BibRecord: BibEntity: Identifiers: – Type: doi Value: 10.1016/j.jbc.2025.110610 Languages: – Code: eng Text: English PhysicalDescription: Pagination: PageCount: 20 StartPage: 1 Subjects: – SubjectFull: Genetic transcription Type: general – SubjectFull: RNA synthesis Type: general – SubjectFull: RNA polymerases Type: general – SubjectFull: Promoters (Genetics) Type: general – SubjectFull: Genetic transcription regulation Type: general Titles: – TitleFull: Quantitative parameters of productive transcription on T5 N25–based promoters are modulated by the initial transcribed sequence and template supercoiling. Type: main BibRelationships: HasContributorRelationships: – PersonEntity: Name: NameFull: Hsu, Lilian M. – PersonEntity: Name: NameFull: Han, N. Natalie IsPartOfRelationships: – BibEntity: Dates: – D: 01 M: 10 Text: Oct2025 Type: published Y: 2025 Identifiers: – Type: issn-print Value: 00219258 Numbering: – Type: volume Value: 301 – Type: issue Value: 10 Titles: – TitleFull: Journal of Biological Chemistry Type: main |
| ResultId | 1 |
