A Modified Yeast-one Hybrid System for Heteromeric Protein Complex-DNA Interaction Studies

Summary

The modified yeast one-hybrid assay described here is an extension of the classical yeast one-hybrid (Y1H) assay to study and validate the heteromeric protein complex-DNA interaction in a heterologous system for any functional genomics study.

Abstract

Over the years, the yeast one-hybrid assay has proven to be an important technique for the identification and validation of physical interactions between proteins such as transcription factors (TFs) and their DNA target. The method presented here utilizes the underlying concept of the Y1H but is modified further to study and validate protein complexes binding to their target DNA. Hence, it is referred to as the modified yeast one-hybrid (Y1.5H) assay. This assay is cost effective and can be easily performed in a regular laboratory setting. Albeit using a heterologous system, the described method could be a valuable tool to test and validate the heteromeric protein complex binding to their DNA target(s) for functional genomics in any system of study, especially plant genomics.

Introduction

In general, to understand protein-DNA interactions, the Y1H assay is the preferred system successfully used in a laboratory setting ¹. The basic Y1H assay involves two components: a) a reporter construct with DNA of interest successfully cloned upstream of a gene encoding a reporter protein; and b) an expression construct which will generate a fusion protein between the TF of interest and a yeast transcription activation domain (AD). The DNA of interest is commonly referred to as 'bait' while the fusion protein is known as 'prey'. In past years, multiple versions of the Y1H assay have been developed to suit specific needs with t....

Protocol

1. Construct and Reporter Plasmid Preparation

1. PCR amplify the promoter regions / "fragments" (preferably ~250 - 500 bp) of the target DNA to be analyzed for further cloning into the entry vector using E. coli (TOP10).
2. Upon sequencing confirmation, subclone the positive clone in a compatible pGLacZi expression vector ⁷ as previously described ^8,9,10,11.
3. Transform the yeast strain for promoter integration (YM4271) by homologous recombination of pGLacZi to generate yeast r....

Results

The general plate set-up procedure for the co-transformation of DNA regions in the yeast cells with protein of interest (Figure 2). The plate set-up can be modified according to the need of the experiment and number of DNA regions/fragments tested. After day-5 of the protocol a well-grown and positive plate should be visible as in Figure 3. In our study, the promoter region of 2600 bp upstream from the start of the transcription .......

Discussion

The existing Y1H standard procedure is suitable for identifying a single protein prey binding to its DNA bait. With various technical modifications, the existing system has been harnessed for defining transcriptional regulatory networks. However, TFs are known to function as a part of complexes involving two or more TFs or proteins, with only some of the TF capable of binding to the DNA. Proteins or TFs which possess only the protein-binding domains and lack the ability to bind to DNA are more likely to work in a complex.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
pDEST22 vector	Invitrogen	PQ1000101	Pro-Quest Two hybrid system kit
pDEST32delatDBD	Invitrogen	PQ1000102	Pro-Quest Two hybrid system kit; this vecor is modified. It is pDEST32 minus DNA binding Domain
pENTR/D-TOPO Cloning Kit	Invitrogen	K240020
YM4271 Strain	Clontech	K1603-1	MATCHMAKER One-Hybrid System
pEXP-AD502	Invitrogen	PQ1000101	Pro-Quest Two hybrid system kit
GATEWAY LR Clonase II enzyme mix	Invitrogen	11791020
YPDA media	Clontech	630410
SD-Agar	Clontech	630412
SD minimal media	Clontech	630411
Uracil DO Supplement	Clontech	630416
Tryptophan Do Supplement	Clontech	630413
Tris Base	Fisher Scientific	BP152-1
EDTA	Fisher Scientific	S311-500
LiAc	Sigma-Aldrich	L4158
Saplmon Sperm (10mg/ml)	Invitrogen	15632011
96 well round bottom Plate	Greiner bio-one	650101
PEG3350	Sigma-Aldrich	1546547
96-deep well block	USA Scientific	1896-2000
Sealable Foil	USA Scientific	2923-0110
Araseal	Excel Scientific	B-100
2-mercaptoethanol	Fisher Scientific	034461-100
ONPG	Sigma-Aldrich	73660
Na2CO3	Sigma-Aldrich	223484
Na2HPO4	Sigma-Aldrich	S3264
NaH2PO4	Sigma-Aldrich	S3139
KCL	Fisher Scientific	BP366-500
MgSO4	Sigma-Aldrich	83266
HCL	Fisher Scientific	SA54-4
Drybath	Thermo Fischer
Voretx	Thermo Fischer
Centrifgue	Eppendorf	Centrifuge 5810R
Plate Reader	Molecular Device		SPECTRAMAX PLUS Microplate Spectrophotometer
Incubator	Thermo Fisher	Model No. 5250- 37 Degree, 6250-30 degrees
Shaker Incubator	New Brunswick
Water Bath	Thermo Fisher	IsoTemp 205
Puncher
50 ml Falocn	BD Falcon
Beaker	Nalgene
Flask	Nalgene
Petriplates (150mm)	Greiner bio-one