Workflow to Select Functional Promoter DNA Baits and Screen Arrayed Gene Libraries in Yeast

The yeast one-hybrid system (Y1H) is used extensively to identify DNA-protein interactions. The generation of large collections of open reading frames (ORFs) to be used as prey in screenings is not a bottleneck nowadays and can be carried out in-house or offered as a service by companies. However, the straightforward use of full gene promoters as baits to identify interacting proteins undermines the accuracy and sensitivity of the assay, especially in the case of multicellular eukaryotes. Therefore, it is paramount to implement procedures for efficient identification of suitable promoter fragments compatible with the Y1H assay. Here, we describe a workflow to identify biologically relevant conserved promoter fragments of Arabidopsis thaliana through simple and robust phylogenetic analyses. Additionally, we describe a manual method and its automated robotized version for rapid and efficient high-throughput Y1H screenings of arrayed ORF libraries with the identified DNA fragments. Moreover, this method can be scaled up or down and used for yeast two-hybrid screenings to search for possible interactors of proteins identified by the Y1H approach or any other protein of interest, altogether underscoring its suitability to build gene regulatory networks. © 2024 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Selection of DNA baits for Y1H screenings Basic Protocol 2: Y1H screenings with arrayed gene libraries Alternate Protocol: Automated screening with a liquid-handling robot.

The work in L.O.-S's lab is supported with grants PID2019-109154RB-I00 and PID2022-142059OB-I00 funded by Spanish MCIN/AEI/ 10.13039/501100011033 and MCIN/AEI/10.13039/5011000 11033/FEDER, UE, respectively. IF-P is supported by a PhD contract (FPI) funded by grant SEV-2016-0672 to the CBGP (Centre of Excellence Severo Ochoa program of the Agencia Estatal de Investigación, Spain). The work in A.G.-M's lab is supported by the ECCO (ERC-2021-COG-101044360) Contract of the EU and grants MULTI-SYSBIO (PID2020-117205GA-I00), BIOELECTRIC (CNS2022-135951), and MULTI-SYNBIO (PID2023-152470NB-I00) funded by MICIU/AEI/ 10.13039/501100011033.

With funding from the Spanish government through the ‘Severo Ochoa Centre of Excellence’ accreditation (SEV-2016-0672)

Peer reviewed