Protocol

Riboswitches

Volume 540 of the series Methods in Molecular Biology pp 215-232

Date:

Probing mRNA Structure and sRNA–mRNA Interactions in Bacteria Using Enzymes and Lead(II)

  • Clément ChevalierAffiliated withArchitecture et Réactivité de l’ARN, Université de Strasbourg
  • , Thomas GeissmannAffiliated withArchitecture et Réactivité de l’ARN, Université de Strasbourg
  • , Anne-Catherine HelferAffiliated withArchitecture et Réactivité de l’ARN, Université de Strasbourg
  • , Pascale RombyAffiliated withArchitecture et Réactivité de l’ARN, Université de Strasbourg

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Summary

Enzymatic probing and lead(II)-induced cleavages have been developed to study the secondary structure of RNA molecules either free or engaged in complex with different ligands. Using a combination of probes with different specificities (unpaired vs. paired regions), it is possible to get information on the accessibility of each nucleotide, on the binding site of a ligand (noncoding RNAs, protein, metabolites), and on RNA conformational changes that accompanied ligand binding or environmental conditions (temperature, pH, ions, etc.). The detection of the cleavages can be conducted by two different ways, which are chosen according to the length of the studied RNA. The first method uses end-labeled RNA molecules and the second one involves primer extension by reverse transcriptase. We provide here an experimental procedure that was designed to map the structure of mRNA and mRNA–sRNA interaction in vitro.

Key words:

RNA RNA–RNA interaction Secondary structure RNA structure probing Ribonuclease Lead(II)-induced cleavages