Investigation of sRNA-mRNA Interactions in Bacillus subtilis In Vivo
© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature..
In this chapter, we describe in vivo methods for the analysis of interactions between an sRNA and its target mRNA in B. subtilis. All these methods have been either established or significantly improved in our group and successfully employed to characterize a number of sRNA/target mRNA systems in Bacillus subtilis. Whereas in Chap. 8, we describe a combination of in vitro methods, e.g., EMSA and RNA secondary structure probing, we focus here on the investigation of RNA-RNA interactions in vivo using compatible plasmids or chromosomal insertions and deletions, the elucidation of the mechanisms of action of regulatory sRNAs employing transcriptional and translational reporter gene fusions, as well as the determination of expression profiles, half-lives of sRNA and mRNA, and their intracellular concentrations, and, finally, the investigation of RNA chaperones that promote the sRNA/mRNA interaction. For an in-depth analysis of sRNA-mRNA interactions in B. subtilis, a combination of in vivo and in vitro methods should be applied.
| Medienart: |
E-Artikel |
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| Erscheinungsjahr: |
2024 |
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| Erschienen: |
2024 |
| Enthalten in: |
Zur Gesamtaufnahme - volume:2741 |
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| Enthalten in: |
Methods in molecular biology (Clifton, N.J.) - 2741(2024) vom: 13., Seite 117-144 |
| Sprache: |
Englisch |
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| Beteiligte Personen: |
Ul Haq, Inam [VerfasserIn] |
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| Links: |
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| Themen: |
Compensatory mutations |
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| Anmerkungen: |
Date Completed 15.01.2024 Date Revised 15.01.2024 published: Print Citation Status MEDLINE |
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| doi: |
10.1007/978-1-0716-3565-0_7 |
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| funding: |
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| Förderinstitution / Projekttitel: |
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| 520 | |a In this chapter, we describe in vivo methods for the analysis of interactions between an sRNA and its target mRNA in B. subtilis. All these methods have been either established or significantly improved in our group and successfully employed to characterize a number of sRNA/target mRNA systems in Bacillus subtilis. Whereas in Chap. 8, we describe a combination of in vitro methods, e.g., EMSA and RNA secondary structure probing, we focus here on the investigation of RNA-RNA interactions in vivo using compatible plasmids or chromosomal insertions and deletions, the elucidation of the mechanisms of action of regulatory sRNAs employing transcriptional and translational reporter gene fusions, as well as the determination of expression profiles, half-lives of sRNA and mRNA, and their intracellular concentrations, and, finally, the investigation of RNA chaperones that promote the sRNA/mRNA interaction. For an in-depth analysis of sRNA-mRNA interactions in B. subtilis, a combination of in vivo and in vitro methods should be applied | ||
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