Identification of DNA-binding proteins and protein-protein interactions by yeast one-hybrid and yeast two-hybrid screen.
The regulation of gene activity is a crucial factor in coordinating development, growth and acclimation to environmental changes. By this means, metabolic processes are adjusted according to cellular needs by changing gene expression patterns. In the genome of the model plant Arabidopsis thaliana, more than 7% of the genes are estimated to encode proteins directly involved in gene regulation. Transcription factors (TFs) are able to bind to specific DNA motifs named cis-elements and control the expression of target genes. The regulation may be either activation, stimulation, inhibition or suppression. The activation of genes is mediated by well-coordinated protein-protein interactions between transcription factors and a various number of cofactors. The gene activation networks are still poorly understood. In order to address the involved protein-DNA and protein-protein interactions, a number of methods have been developed that efficiently address cis-element interacting partners. This chapter describes two powerful methods: the yeast one-hybrid system and the yeast two-hybrid system. In combination these techniques provide the ability to identify cis-element-binding transcription factors and their upstream interaction partners.
Klein
Peter
P
Biochemistry and Physiology of Plants, Faculty of Biology, Bielefeld University, Bielefeld, Germany.
Dietz
Karl-Josef
KJ
D017360:Arabidopsis / Q000378:metabolism; D003001:Cloning, Molecular; D004268:DNA-Binding Proteins / Q000378:metabolism*; D015723:Gene Library; D005800:Genes, Fungal / Q000235:genetics; D017930:Genes, Reporter; D011485:Protein Binding; D011995:Recombination, Genetic / Q000235:genetics; D015203:Reproducibility of Results; D012441:Saccharomyces cerevisiae / Q000166:cytology / Q000235:genetics; D014170:Transformation, Genetic; D020798:Two-Hybrid System Techniques
D004268:DNA-Binding Proteins