Riboswitches pp 215-232

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

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

  • Clément Chevalier
  • Thomas Geissmann
  • Anne-Catherine Helfer
  • Pascale Romby
Protocol

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 

References

  1. 1.
    Kaczanowska, M., and Ryden-Aulin, M. (2007). Ribosome biogenesis and the translation process in Escherichia coli. Microbiol. Mol. Biol. Rev. 71, 477–494.PubMedCrossRefGoogle Scholar
  2. 2.
    Nguyenle, T., Laurberg, M., Brenowitz, M., and Noller, H. F. (2006). Following the dynamics of changes in solvent accessibility of 16 S and 23 S rRNA during ribosomal subunit association using synchrotron-generated hydroxyl radicals. J. Mol. Biol. 359, 1235–1248.PubMedCrossRefGoogle Scholar
  3. 3.
    Narberhaus, F., Waldminghaus, T., and Chowdhury, S. (2006). RNA thermometers. FEMS Microbiol. Rev. 30, 3–16.PubMedCrossRefGoogle Scholar
  4. 4.
    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
  5. 5.
    Barrick, J. E., and Breaker, R. R. (2007). The distributions, mechanisms, and structures of metabolite-binding riboswitches. Genome Biol. 8, R239.PubMedCrossRefGoogle Scholar
  6. 6.
    Gottesman, S., McCullen, C. A., Guillier, M., Vanderpool, C. K., Majdalani, N., Benhammou, J., Thompson, K. M., FitzGerald, P. C., Sowa, N. A., and FitzGerald, D. J. (2006). Small RNA regulators and the bacterial response to stress. Cold Spring Harb. Symp. Quant. Biol. 71, 1–11.PubMedCrossRefGoogle Scholar
  7. 7.
    Storz, G., Altuvia, S., and Wassarman, K. M. (2005). An abundance of RNA regulators. Annu. Rev. Biochem. 74, 199–217.PubMedCrossRefGoogle Scholar
  8. 8.
    Wagner, E. G. H., Altuvia, S., and Romby, P. (2002). Antisense RNAs in bacteria and their genetic elements. Adv. Genet. 46, 361–398.PubMedCrossRefGoogle Scholar
  9. 9.
    Brantl, S. (2007). Regulatory mechanisms employed by cis-encoded antisense RNAs. Curr. Opin. Microbiol. 10, 102–109.PubMedCrossRefGoogle Scholar
  10. 10.
    Chen, Y., and Varani, G. (2005). Protein families and RNA recognition. FEBS J. 272, 2088–2097.PubMedCrossRefGoogle Scholar
  11. 11.
    Leontis, N. B., Lescoute, A., and Westhof, E. (2006). The building blocks and motifs of RNA architecture. Curr. Opin. Struct. Biol. 16, 279–287.PubMedCrossRefGoogle Scholar
  12. 12.
    Jossinet, F., Ludwig, T. E., and Westhof, E. (2007). RNA structure: bioinformatic analysis. Curr. Opin. Microbiol. 10, 279–285.PubMedCrossRefGoogle Scholar
  13. 13.
    Zuker, M. (2003). Mfold web server for nucleic acid folding and hybridization prediction. Nucleic Acids Res. 31, 3406–3415.PubMedCrossRefGoogle Scholar
  14. 14.
    Do, C. B., Woods, D. A., and Batzoglou, S. (2006). CONTRAfold: RNA secondary structure prediction without physics-based models. Bioinformatics 22, e90–e98.PubMedCrossRefGoogle Scholar
  15. 15.
    Xayaphoummine, A., Bucher, T., and Isambert, H. (2005). Kinefold web server for RNA/DNA folding path and structure prediction including pseudoknots and knots. Nucleic Acids Res. 33, W605–W610.PubMedCrossRefGoogle Scholar
  16. 16.
    Gultyaev, A. P., van Batenburg, F. H., and Pleij, C. W. (1995). The computer simulation of RNA folding pathways using a genetic algorithm. J. Mol. Biol. 250, 37–51.PubMedCrossRefGoogle Scholar
  17. 17.
    Donis-Keller, H., Maxam, A. M., and Gilbert, W. (1977). Mapping adenines, guanines, and pyrimidines in RNA. Nucleic Acids Res. 4, 2527–2538.PubMedCrossRefGoogle Scholar
  18. 18.
    Ehresmann, C., Baudin, F., Mougel, M., Romby, P., Ebel, J. P., and Ehresmann, B. (1987). Probing the structure of RNAs in solution. Nucleic Acids Res. 15, 9109–9128.PubMedCrossRefGoogle Scholar
  19. 19.
    Lockard, R. E., and Kumar, A. (1981). Mapping tRNA structure in solution using double-strand-specific ribonuclease V1 from cobra venom. Nucleic Acids Res. 9, 5125–5140.PubMedCrossRefGoogle Scholar
  20. 20.
    Favorova, O. O., Fasiolo, F., Keith, G., Vassilenko, S. K., and Ebel, J. P. (1981). Partial digestion of tRNA – aminoacyl-tRNA synthetase complexes with cobra venom ribonuclease. Biochemistry 20, 1006–1011.PubMedCrossRefGoogle Scholar
  21. 21.
    Kolb, F. A., Malmgren, C., Westhof, E., Ehresmann, C., Ehresmann, B., Wagner, E. G. H., and Romby, P. (2000). An unusual structure formed by antisense-target RNA binding involves an extended kissing complex with a four-way junction and a side-by-side helical alignment. RNA 6, 311–324.PubMedCrossRefGoogle Scholar
  22. 22.
    Huntzinger, E., Boisset, S., Saveanu, C., Benito, Y., Geissmann, T., Namane, A., Lina, G., Etienne, J., Ehresmann, B., Ehresmann, C., Jacquier, A., Vandenesch, F., and Romby, P. (2005). Staphylococcus aureus RNAIII and the endoribonuclease III coordinately regulate spa gene expression. EMBO J. 24, 824–835.PubMedCrossRefGoogle Scholar
  23. 23.
    Darfeuille, F., Unoson, C., Vogel, J., and Wagner, E. G. H. (2007) An antisense RNA inhibits translation by competing with standby ribosomes. Mol. Cell 26, 381–392.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.
    Lindell, M., Romby, P., and Wagner, E. G. H. (2002). Lead(II) as a probe for investigating RNA structure in vivo. RNA 8, 534–541.PubMedCrossRefGoogle Scholar
  26. 26.
    Ivanova, N., Lindell, M., Pavlov, M., Holmberg Schiavone, L., Wagner, E. G. H., and Ehrenberg, M. (2007). Structure probing of tmRNA in distinct stages of trans-translation. RNA 13, 713–722.PubMedCrossRefGoogle Scholar
  27. 27.
    Huntzinger, E., Possedko, M., Winter, F., Moine, H., Ehresmann, C., and Romby, P. (2005). Probing RNA structures with enzymes and chemicals in vitro and in vivo, in Handbook of RNA Chemistry (Hartmann, R. K., Bindereif, A., Schön, A., and Westhof, E., eds), Wiley-VCH, Weinheim, pp. 151–171.CrossRefGoogle Scholar
  28. 28.
    Marchand, V., Mougin, A., Méreau, A., and Branlant, C. (2005). Study of RNA–protein interactions and RNA structure in ribonucleoprotein particles, in Handbook of RNA Chemistry (Hartmann, R. K., Bindereif, A., Schön, A., and Westhof, E., eds), Wiley-VCH, Weinheim, pp. 172–228.CrossRefGoogle Scholar
  29. 29.
    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
  30. 30.
    Coppins, R. L., Hall, K. B., and Groisman, E. A. (2007). The intricate world of riboswitches. Curr. Opin. Microbiol. 10, 176–181.PubMedCrossRefGoogle Scholar
  31. 31.
    Fabbretti, A., Pon, C. L., Hennelly, S. P., Hill, W. E., Lodmell, J. S., and Gualerzi, C. O. (2007). The real-time path of translation factor IF3 onto and off the ribosome. Mol. Cell 25, 285–296.PubMedCrossRefGoogle Scholar
  32. 32.
    Shcherbakova, I., Mitra, S., Beer, R. H., and Brenowitz, M. (2006). Fast Fenton footprinting: a laboratory-based method for the time-resolved analysis of DNA, RNA and proteins. Nucleic Acids Res. 34, e48.PubMedCrossRefGoogle Scholar
  33. 33.
    Fabbretti, A., Milon, P., Giuliodori, A. M., Gualerzi, C. O., and Pon, C. L. (2007). Real-time dynamics of ribosome–ligand interaction by time-resolved chemical probing methods. Methods Enzymol. 430, 45–58.PubMedCrossRefGoogle Scholar
  34. 34.
    Boisset, S., Geissmann, T., Huntzinger, E., Fechter, P., Bendridi, N., Possedko, M., Chevalier, C., Helfer, A. C., Benito, Y., Jacquier, A., Gaspin, C., Vandenesch, F., and Romby, P. (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
  35. 35.
    Novick, R. P. (2003). Autoinduction and signal transduction in the regulation of staphylococcal virulence. Mol. Microbiol. 48, 1429–1449.PubMedCrossRefGoogle Scholar
  36. 36.
    Toledo-Arana, A., Repoila, F., and Cossart, P. (2007). Small noncoding RNAs controlling pathogenesis. Curr. Opin. Microbiol. 10, 182–188.PubMedCrossRefGoogle Scholar
  37. 37.
    Kolb, F. A., Engdahl, H. M., Slagter-Jäger, J. G., Ehresmann, B., Ehresmann, C., Westhof, E., Wagner, E. G. H., and Romby, P. (2000). Progression of a loop–loop complex to a four-way junction is crucial for the activity of a regulatory antisense RNA. EMBO J. 19, 5905–5915.PubMedCrossRefGoogle Scholar
  38. 38.
    Qu, H. L., Michot, B., and Bachellerie, J. P. (1983). Improved methods for structure probing in large RNAs: a rapid ‘heterologous’ sequencing approach is coupled to the direct mapping of nuclease accessible sites. Application to the 5′ terminal domain of eukaryotic 28S rRNA. Nucleic Acids Res. 11, 5903–5920.PubMedCrossRefGoogle Scholar
  39. 39.
    Milligan, J. F., and Uhlenbeck, O. C. (1989). Synthesis of small RNAs using T7 RNA polymerase. Methods Enzymol. 180, 51–62.PubMedCrossRefGoogle Scholar
  40. 40.
    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
  41. 41.
    Jahn, M. J., Jahn, D., Kumar, A. M., and Söll, 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
  42. 42.
    England, T. E., Bruce, A. G., and Uhlenbeck, O. C. (1980). Specific labeling of 3′ termini of RNA with T4 RNA ligase. Methods Enzymol. 65, 65–74.CrossRefGoogle Scholar
  43. 43.
    Walker, S. C., Avis, J. M., and Conn, G. L. (2003). General plasmids for producing RNA in vitro transcripts with homogeneous ends. Nucleic Acids Res. 31, e82.PubMedCrossRefGoogle Scholar

Copyright information

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

Authors and Affiliations

  • Clément Chevalier
    • 1
  • Thomas Geissmann
    • 1
  • Anne-Catherine Helfer
    • 1
  • Pascale Romby
    • 1
  1. 1.Architecture et Réactivité de l’ARNUniversité de StrasbourgFrance

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