Protocol

Riboswitches

Volume 540 of the series Methods in Molecular Biology pp 247-263

Date:

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

  • Pierre FechterAffiliated withArchitecture et Réactivité de l’ARN, Université de Strasbourg
  • , Clément ChevalierAffiliated withArchitecture et Réactivité de l’ARN, Université de Strasbourg
  • , Gulnara YusupovaAffiliated withArchitecture et Réactivité de l’ARN, Université de Strasbourg
  • , Marat YusupovAffiliated withArchitecture et Réactivité de l’ARN, Université de Strasbourg
  • , Pascale RombyAffiliated withArchitecture et Réactivité de l’ARN, Université de Strasbourg
  • , Stefano MarziAffiliated withArchitecture et Réactivité de l’ARN, Université de Strasbourg

* Final gross prices may vary according to local VAT.

Get Access

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