Ohuchi, Shoji ; Mascher, Thorsten ; Suess, Beatrix (2019)
Promoter RNA sequencing (PRSeq) for the massive and quantitative promoter analysis in vitro.
In: Scientific reports, 9 (1)
doi: 10.1038/s41598-019-39892-x
Artikel, Bibliographie
Kurzbeschreibung (Abstract)
Analysis of promoter strength and specificity is important for understanding and engineering gene regulation. Here, we report an in vitro promoter analysis method that can achieve both massiveness and quantitativeness. In this approach, a pool of single-stranded DNA with a partially randomized promoter sequence to be analyzed is chemically synthesized. Through enzymatic reactions, the randomized sequence will be copied to the downstream region, resulting in a template DNA pool that carries its own promoter information on its transcribed region. After in vitro transcription of the DNA pool with an RNA polymerase of interest, the sequences of the resulting transcripts will be analyzed. Since the promoter strength linearly correlates to the copy number of transcript, the strength of each promoter sequence can be evaluated. A model experiment of T7 promoter variants demonstrated the quantitativeness of the method, and the method was applied for the analysis of the promoter of cyanophage Syn5 RNA polymerase. This method provides a powerful approach for analyzing the complexity of promoter specificity and discrimination for highly abundant and often redundant alternative sigma factors such as the extracellular function (ECF) sigma factors.
| Typ des Eintrags: | Artikel |
|---|---|
| Erschienen: | 2019 |
| Autor(en): | Ohuchi, Shoji ; Mascher, Thorsten ; Suess, Beatrix |
| Art des Eintrags: | Bibliographie |
| Titel: | Promoter RNA sequencing (PRSeq) for the massive and quantitative promoter analysis in vitro. |
| Sprache: | Englisch |
| Publikationsjahr: | 28 Februar 2019 |
| Titel der Zeitschrift, Zeitung oder Schriftenreihe: | Scientific reports |
| Jahrgang/Volume einer Zeitschrift: | 9 |
| (Heft-)Nummer: | 1 |
| DOI: | 10.1038/s41598-019-39892-x |
| Kurzbeschreibung (Abstract): | Analysis of promoter strength and specificity is important for understanding and engineering gene regulation. Here, we report an in vitro promoter analysis method that can achieve both massiveness and quantitativeness. In this approach, a pool of single-stranded DNA with a partially randomized promoter sequence to be analyzed is chemically synthesized. Through enzymatic reactions, the randomized sequence will be copied to the downstream region, resulting in a template DNA pool that carries its own promoter information on its transcribed region. After in vitro transcription of the DNA pool with an RNA polymerase of interest, the sequences of the resulting transcripts will be analyzed. Since the promoter strength linearly correlates to the copy number of transcript, the strength of each promoter sequence can be evaluated. A model experiment of T7 promoter variants demonstrated the quantitativeness of the method, and the method was applied for the analysis of the promoter of cyanophage Syn5 RNA polymerase. This method provides a powerful approach for analyzing the complexity of promoter specificity and discrimination for highly abundant and often redundant alternative sigma factors such as the extracellular function (ECF) sigma factors. |
| ID-Nummer: | pmid:30816266 |
| Fachbereich(e)/-gebiet(e): | 10 Fachbereich Biologie 10 Fachbereich Biologie > Synthetic Genetic Circuits (2020 umbenannt in "Synthetic RNA biology") |
| Hinterlegungsdatum: | 05 Mär 2019 06:40 |
| Letzte Änderung: | 05 Mär 2019 06:40 |
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