Isolation of Proteins Binding to Promoter Elements of Alkaloid Metabolism-Related Genes Using Yeast One-Hybrid

  • Débora Vom Endt
  • Giancarlo Pasquali
Part of the Methods in Molecular Biology book series (MIMB, volume 643)


Controlled transcription of biosynthetic genes is one major mechanism regulating alkaloid production in plant cells. This regulation of biosynthetic pathways is achieved by specific transcription factors. Sequence-specific DNA-binding proteins interact with the promoter regions of target genes, modulating the rate of initiation of mRNA synthesis by RNA polymerase II. Gene transcription is regulated depending on tissue type and/or in response to internal signals like hormones or external signals such as microbial elicitors or UV light. Promoter elements are identified based on their ability to keep the wild-type response to these signals. Transcription factors involved in biosynthetic regulation can be isolated based on their ability to bind these specific promoter elements using yeast one-hybrid screening. Several transcription factors involved in the regulation of alkaloid metabolism-related genes have been isolated by this method. The aim of this chapter is to describe the yeast one-hybrid system for screening DNA-binding proteins potentially involved in transcriptional regulation.

Key words

Yeast one-hybrid transcription factor alkaloid metabolism DNA-binding protein promoter element library screening 



Authors are grateful to Dr. Luís F. Revers (Embrapa Uva and Vinho, Bento Gonçalves, RS, Brazil) and Danielle Costenaro da Silva, M.Sc. (PPGBCM/CBiot, UFRGS, Porto Alegre, RS, Brazil), for Fig. 5.3; Dr. Jacqueline M. Cardone (Exponencial Biotecnologia Ltd., Porto Alegre, RS, Brazil) for advice and careful reading of draft versions.


  1. 1.
    Menke, F. L. H., Champion, A., Kijne, J. W., and Memelink, J. (1999) A novel jasmonate- and elicitor-responsive element in the periwinkle secondary metabolite biosynthetic gene Str interacts with a jasmonate- and elicitor-inducible AP2-domain transcription factor, ORCA2. EMBO J. 18, 4455–4463.PubMedCrossRefGoogle Scholar
  2. 2.
    Pré, M., Sibéril, Y., Memelink, J., Champion, A., Doireau, P., and Gantet, P. (2000) Isolation by the yeast one-hybrid system of cDNAs encoding transcription factors that bind to the G-box element of the strictosidine synthase gene promoter from Catharanthus roseus. Int. J. Bio-Chromatogr. 5, 229–244.Google Scholar
  3. 3.
    van der Fits, L., Zhang, H., Menke, F. L. H., Deneka, M., and Memelink, J. (2000) A Catharanthus roseus BPF-1 homologue interacts with an elicitor-responsive region of the secondary metabolite biosynthetic gene Str and is induced by elicitor via a jasmonate independent signal transduction pathway. Plant Mol. Biol. 44, 675–685.PubMedCrossRefGoogle Scholar
  4. 4.
    Vom Endt, D., Soares e Silva, M., Kijne, J. W., Pasquali, G., and Memelink, J. (2007) Identification of a bipartite jasmonate-responsive promoter element in the Catharanthus roseus ORCA3 transcription factor gene that interacts specifically with AT-Hook DNA-binding proteins. Plant Physiol. 144, 1680–1689.CrossRefGoogle Scholar
  5. 5.
    Clontech Laboratories Inc. (2007) Matchmaker library construction & screening kits user manual. (accessed August 31, 2009).
  6. 6.
    Marsh, J. L., Erfle, M., and Wykes, E. J. (1984) The pIC plasmid and phage vectors with versatile cloning sites for recombinant selection by insertional inactivation. Gene 32, 481–485.PubMedCrossRefGoogle Scholar
  7. 7.
    Meijer, A. H., Ouwerkerk, P. B. F., and Hoge, J. H. C. (1998) Vectors for transcription factor cloning and target site identification by means of genetic selection in yeast. Yeast 14, 1407–1416.PubMedCrossRefGoogle Scholar
  8. 8.
    Ziegler, J., and Facchini, P. J. (2008) Alkaloid biosynthesis: metabolism and trafficking. Annu. Rev. Plant Biol. 59, 735–769.PubMedCrossRefGoogle Scholar
  9. 9.
    Ouwerkerk, P. B. F., and Memelink, J. (1997) A simple method for directional multimerization of DNA sequences. Trends Genet. 13, 207.CrossRefGoogle Scholar
  10. 10.
    Zhu, Y. Y., Machleder, E.M., Chenchik, A., Li, R., and Siebert, P. D. (2001) Reverse transcriptase template switching, a SMART approach for full-length cDNA library construction. Biotechniques 30, 892–897.PubMedGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2010

Authors and Affiliations

  • Débora Vom Endt
    • 1
  • Giancarlo Pasquali
    • 2
  1. 1.State University of Rio Grande do SulNovo HamburgoBrazil
  2. 2.Center for BiotechnologyFederal University of Rio Grande do SulPorto AlegreBrazil

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