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