Summary
Toeprinting was developed to study the formation of ribosomal initiation complexes in bacteria. This approach, based on the inhibition of reverse transcriptase elongation, was used to monitor the effect of ribosomal components and translational factors on the formation of the active ribosomal initiation complex. Moreover, this method offers an easy way to study in vitro how mRNA conformational changes alter ribosome binding at the initiation site. These changes can be induced either by environmental cues (temperature, ion concentration), or by the binding of metabolites, regulatory proteins, and trans-acting RNAs. An experimental guide is given to follow the different steps of the formation of ribosomal initiation complexes in Escherichia coli and Staphylococcus aureus, and to monitor the mechanism of action of several regulators on translation initiation in vitro. Protocols to prepare the ribosome and the subunits are also given for Thermus thermophilus, Staphylococcus aureus, and Escherichia coli.
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Acknowledgments
We are grateful to Anne-Catherine Helfer for the toeprinting assays performed on E. coli rpsO mRNA. This work was supported by financial support from the Centre National de la Recherche Scientifique (UPR 9002 CNRS), from the University Louis Pasteur of Strasbourg, from the Ministère de la Recherche (ANR05-MIIME, ANR07-BLANC), from the European Community (FOSRAK, EC005120; BacRNA EC018618).
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Fechter, P., Chevalier, C., Yusupova, G., Yusupov, M., Romby, P., Marzi, S. (2009). Ribosomal Initiation Complexes Probed by Toeprinting and Effect of trans-Acting Translational Regulators in Bacteria . In: Serganov, A. (eds) Riboswitches. Methods in Molecular Biology, vol 540. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-59745-558-9_18
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DOI: https://doi.org/10.1007/978-1-59745-558-9_18
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