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


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 


  1. 1.
    Serganov, A. and Patel, D. J. (2007). Ribozymes, riboswitches and beyond: regulation of gene expression without proteins. Nat. Rev. Genet. 8, 776–790.PubMedCrossRefGoogle Scholar
  2. 2.
    Romby, P. and Springer, M. (2007). Translational control in prokaryotes. In Translational Control in Biology and Medicine (Hershey, J, Sonnenberg, N, Metthews, M, eds), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, pp 807–832.Google Scholar
  3. 3.
    Narberhaus, F., Waldminghaus, T. and Chowdhury, S. (2006). RNA thermometers. FEMS Microbiol. Rev. 30, 3–16.PubMedCrossRefGoogle Scholar
  4. 4.
    Coppins, R. L., Hall, K. B. and Groisman, E. A. (2007). The intricate world of riboswitches. Curr. Opin. Microbiol. 10, 176–181.PubMedCrossRefGoogle Scholar
  5. 5.
    Romby, P. and Springer, M. (2003). Bacterial translational control at atomic resolution. Trends Genet. 19, 155–161.PubMedCrossRefGoogle Scholar
  6. 6.
    Schlax, P. J. and Worhunsky, D. J. (2003). Translational repression mechanisms in prokaryotes. Mol. Microbiol. 48, 1157–1169.PubMedCrossRefGoogle Scholar
  7. 7.
    Ehresmann, C., Ehresmann, B., Ennifar, E., Dumas, P., Garber, M., Mathy, N. et al (2004). Molecular mimicry in translational regulation: the case of ribosomal protein S15. RNA Biol. 1, 66–73.PubMedCrossRefGoogle Scholar
  8. 8.
    Marzi, S., Myasnikov, A. G., Serganov, A., Ehresmann, C., Romby, P., Yusupov, M. et al (2007). Structured mRNAs regulate translation initiation by binding to the platform of the ribosome. Cell 130, 1019–1031.PubMedCrossRefGoogle Scholar
  9. 9.
    Darfeuille, F., Unoson, C., Vogel, J. and Wagner, E. G. (2007). An antisense RNA inhibits translation by competing with standby ribosomes. Mol. Cell 26, 381–392.PubMedCrossRefGoogle Scholar
  10. 10.
    Vogel, J. and Wagner, E. G. (2007). Target identification of small noncoding RNAs in bacteria. Curr. Opin. Microbiol. 10, 262–270.PubMedCrossRefGoogle Scholar
  11. 11.
    Hartz, D., McPheeters, D. S., Traut, R. and Gold, L. (1988). Extension inhibition analysis of translation initiation complexes. Methods Enzymol. 164, 419–425.PubMedCrossRefGoogle Scholar
  12. 12.
    Hartz, D., McPheeters, D. S. and Gold, L. (1989). Selection of the initiator tRNA by Escherichia coli initiation factors. Genes Dev. 3, 1899–1912.PubMedCrossRefGoogle Scholar
  13. 13.
    Huttenhofer, A. and Noller, H. F. (1994). Footprinting mRNA–ribosome complexes with chemical probes. EMBO J. 13, 3892–3901.PubMedGoogle Scholar
  14. 14.
    Sacerdot, C., Caillet, J., Graffe, M., Eyermann, F., Ehresmann, B., Ehresmann, C. et al (1998). The Escherichia coli threonyl-tRNA synthetase gene contains a split ribosomal binding site interrupted by a hairpin structure that is essential for autoregulation. Mol. Microbiol. 29, 1077–1090.PubMedCrossRefGoogle Scholar
  15. 15.
    Yusupova, G. Z., Yusupov, M. M., Cate, J. H. and Noller, H. F. (2001). The path of messenger RNA through the ribosome. Cell 106, 233–241.PubMedCrossRefGoogle Scholar
  16. 16.
    Jenner, L., Romby, P., Rees, B., Schulze-Briese, C., Springer, M., Ehresmann, C. et al (2005). Translational operator of mRNA on the ribosome: how repressor proteins exclude ribosome binding. Science 308, 120–123.PubMedCrossRefGoogle Scholar
  17. 17.
    Korostelev, A., Trakhanov, S., Asahara, H., Laurberg, M., Lancaster, L. and Noller, H. F. (2007). Interactions and dynamics of the Shine Dalgarno helix in the 70S ribosome. Proc. Natl Acad. Sci. U. S. A. 104, 16840–16843.PubMedCrossRefGoogle Scholar
  18. 18.
    Hartz, D., McPheeters, D. S., Green, L. and Gold, L. (1991). Detection of Escherichia coli ribosome binding at translation initiation sites in the absence of tRNA. J. Mol. Biol. 218, 99–105.PubMedCrossRefGoogle Scholar
  19. 19.
    Philippe, C., Eyermann, F., Benard, L., Portier, C., Ehresmann, B. and Ehresmann, C. (1993). Ribosomal protein S15 from Escherichia coli modulates its own translation by trapping the ribosome on the mRNA initiation loading site. Proc. Natl Acad. Sci. U. S. A. 90, 4394–4398.PubMedCrossRefGoogle Scholar
  20. 20.
    Ringquist, S., MacDonald, M., Gibson, T. and Gold, L. (1993). Nature of the ribosomal mRNA track: analysis of ribosome-binding sites containing different sequences and secondary structures. Biochemistry 32, 10254–10262.PubMedCrossRefGoogle Scholar
  21. 21.
    Qin, Y., Polacek, N., Vesper, O., Staub, E., Einfeldt, E., Wilson, D. N. et al (2006). The highly conserved LepA is a ribosomal elongation factor that back-translocates the ribosome. Cell 127, 721–733.PubMedCrossRefGoogle Scholar
  22. 22.
    Waldminghaus, T., Heidrich, N., Brantl, S. and Narberhaus, F. (2007). FourU: a novel type of RNA thermometer in Salmonella. Mol. Microbiol. 65, 413–424.PubMedCrossRefGoogle Scholar
  23. 23.
    Brunel, C., Romby, P., Moine, H., Caillet, J., Grunberg-Manago, M., Springer, M. et al (1993). Translational regulation of the Escherichia coli threonyl-tRNA synthetase gene: structural and functional importance of the thrS operator domains. Biochimie 75, 1167–7119.PubMedCrossRefGoogle Scholar
  24. 24.
    Sharma, C. M., Darfeuille, F., Plantinga, T. H. and Vogel, J. (2007). A small RNA regulates multiple ABC transporter mRNAs by targeting C/A-rich elements inside and upstream of ribosome-binding sites. Genes Dev 21, 2804–2817.PubMedCrossRefGoogle Scholar
  25. 25.
    Boisset, S., Geissmann, T., Huntzinger, E., Fechter, P., Bendridi, N., Possedko, M. et al (2007). Staphylococcus aureus RNAIII coordinately represses the synthesis of virulence factors and the transcription regulator Rot by an antisense mechanism. Genes Dev. 21, 1353–1366.PubMedCrossRefGoogle Scholar
  26. 26.
    Milligan, J. F. and Uhlenbeck, O. C. (1989). Synthesis of small RNAs using T7 RNA polymerase. Methods Enzymol. 180, 51–62.PubMedCrossRefGoogle Scholar
  27. 27.
    Romaniuk, P. J., de Stevenson, I. L. and Wong, H. H. (1987). Defining the binding site of Xenopus transcription factor IIIA on 5S RNA using truncated and chimeric 5S RNA molecules. Nucleic Acids Res. 15, 2737–2755.PubMedCrossRefGoogle Scholar
  28. 28.
    Jahn, M. J., Jahn, D., Kumar, A. M. and Soll, D. (1991). Mono Q chromatography permits recycling of DNA template and purification of RNA transcripts after T7 RNA polymerase reaction. Nucleic Acids Res. 19, 2786.PubMedCrossRefGoogle Scholar
  29. 29.
    Marzi, S., Fechter, P., Chevalier, C., Romby, P. and Geissmann, T. RNA switches regulate initiation of translation in bacteria. Biol. Chem. 389, 585–598.Google Scholar
  30. 30.
    Novick, R. P. and Jiang, D. (2003). The staphylococcal saeRS system coordinates environmental signals with agr quorum sensing. Microbiology 149, 2709–2717.PubMedCrossRefGoogle Scholar

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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