Abstract
So far, in Bacillus subtilis, only four trans-encoded and 11 cis-encoded sRNAs and their targets have been investigated in detail, the majority of them in our group (rev. in 1, 2). Here, we describe in vitro methods for the analysis of sRNA/mRNA interactions. All these methods have been either elaborated or significantly improved in our group and successfully applied to characterize a number of sRNA/target mRNA systems in Bacillus subtilis for which we provide examples from our own work. The in vitro methods comprise the synthesis and purification of labeled and unlabeled RNA, the analysis of sRNA/mRNA interactions in electrophoretic mobility shift assays (EMSAs) including the calculation of their apparent binding rate constants (kapp) and equilibrium dissociation constants (Kd), the localization of minimal regulatory regions of an sRNA, the determination of the secondary structures of both interacting RNAs and their complex as well as the analysis of RNA chaperones that may promote the sRNA/mRNA interaction.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Similar content being viewed by others
References
Heidrich N, Moll I, Brantl S (2007) In vitro analysis of the interaction between the small RNA SR1 and its primary target ahrC mRNA. Nucleic Acids Res 35:43331–44346
Müller P, Gimpel M, Wildenhain T, Brantl S (2019) A new role for CsrA: promotion of complex formation between an sRNA and its mRNA target in Bacillus subtilis. RNA Biol 16:972–987
Brantl S, Wagner EGH (1994) Antisense RNA-mediated transcriptional attenuation occurs faster than stable antisense/target RNA pairing: an in vitro study of plasmid pIP501. EMBO J 13:3599–3607
Jahn N, Brantl S (2013) One antitoxin – two functions: SR4 controls toxin mRNA decay and translation. Nucleic Acids Res 41:9870–9880
Brantl S, Wagner EGH (2000) Antisense-RNA mediated transcriptional attenuation: an in vitro study of plasmid pT181. Mol Microbiol 35:1469–1482
Meißner C, Jahn N, Brantl S (2016) In vitro characterization of the type I toxin-antitoxin system bsrE/SR5 from Bacillus subtilis. J Biol Chem 291:560–571
Ul Haq I, Brantl S, Müller P (2021) A new role for SR1 from Bacillus subtilis: regulation of sporulation by inhibition of kinA translation. Nucleic Acids Res 49:10589–10603
Heidrich N, Brantl S (2003) Antisense-RNA mediated transcriptional attenuation: importance of a U-turn loop structure in the target RNA of plasmid pIP501 for efficient inhibition by the antisense RNA. J Mol Biol 33:917–929
Gimpel M, Brantl S (2016) Dual-function sRNA encoded peptide SR1P modulates moonlighting activity of B. subtilis GapA. RNA Biol 13:916–926
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2024 The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
Ul Haq, I., Müller, P., Brantl, S. (2024). In Vitro Methods for the Investigation of sRNA-mRNA Interactions in Bacillus subtilis. In: Arluison, V., Valverde, C. (eds) Bacterial Regulatory RNA. Methods in Molecular Biology, vol 2741. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-3565-0_8
Download citation
DOI: https://doi.org/10.1007/978-1-0716-3565-0_8
Published:
Publisher Name: Humana, New York, NY
Print ISBN: 978-1-0716-3564-3
Online ISBN: 978-1-0716-3565-0
eBook Packages: Springer Protocols