Riboswitches pp 247-263

Part of the Methods in Molecular Biology book series (MIMB, volume 540)

Ribosomal Initiation Complexes Probed by Toeprinting and Effect of trans-Acting Translational Regulators in Bacteria

  • Pierre Fechter
  • Clément Chevalier
  • Gulnara Yusupova
  • Marat Yusupov
  • Pascale Romby
  • Stefano Marzi
Protocol

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.

Key words:

Ribosome Ribosome purification mRNA Translation initiation Toeprinting Translational regulator Escherichia coli Staphylococcus aureus Thermus thermophilus 

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Copyright information

© Humana Press, a part of Springer Science+Business Media, LLC 2009

Authors and Affiliations

  • Pierre Fechter
    • 1
  • Clément Chevalier
    • 1
  • Gulnara Yusupova
    • 1
  • Marat Yusupov
    • 1
  • Pascale Romby
    • 1
  • Stefano Marzi
    • 1
  1. 1.Architecture et Réactivité de l’ARNUniversité de StrasbourgFrance

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