Protocol

Gateway-Compatible Yeast One-Hybrid Screens

Program in Gene Function and Expression and Program in Molecular Medicine, University of Massachusetts Medical School, Worcester, MA 01605, USA

¹Corresponding author (marian.walhout{at}umassmed.edu)

INTRODUCTION

Protein-DNA interactions (PDIs) between transcription factors (TFs) and their target genes form the backbone of transcription regulatory networks. Such PDIs can be identified with either a TF or a gene as a starting point. The Gateway-compatible yeast one-hybrid (Y1H) system provides a high-throughput, gene-centered method for the identification of interactions between a "DNA bait" (e.g., cis-regulatory DNA elements or gene promoters) and "protein preys" (e.g., TFs). The Y1H system is a genetic system to detect PDIs based on selection of reporter gene expression in yeast. DNA baits are fused by Gateway cloning to two reporter genes, HIS3 and lacZ, and the resulting DNA bait::reporter constructs are subsequently integrated into the genome of the host yeast strain. After integration, baits are examined for self-activation (i.e., their ability to drive reporter gene expression in the absence of an exogenous prey protein). Subsequently, each DNA bait is screened for interacting proteins by transforming a library of preys into the corresponding Y1H DNA bait yeast strain. Preys are hybrid proteins composed of a protein from the organism of interest and a heterologous transcription activation domain. When a prey protein binds to the DNA bait, the heterologous activation domain activates reporter gene expression. Thus, physical interactions between both repressors and activators and their DNA targets can be identified.