Yeast Two-Hybrid SystemA SummaryUpdated July 2016

Purpose:To determine direct protein-protein interactions

Background: Transcription Factors DNA-

binding Domain

Activation

Domain

Upstream

Activation

Sequence

(UAS)

Reporter Gene

RNA Polymerase

Transcription Factor

Protein Fusion

Bait

DNA-binding Domain

Activation

Domain

Transcription Factor

Prey

AB

C

I

D

E

F G

Interaction of Bait and Prey

Bait

DNA-binding Domain

Upstream

Activation

Sequence

(UAS)

Reporter Gene

Prey

Activation

Domain

RNA Polymerase

Recovery of Information

To be sequenced…

Y2H

Pros: Direct interactions Doesn’t require

sophisticated equipment/machinery

Can screen many proteins

Can be automated

Cons: Limited to soluble proteins Addressing transient

nature of interactions False positives Protein fusion could inhibit

some interactions Takes place in nucleus and

proteins localized elsewhere may not show up in screen

Some false positives may not be in the same cell at the same time

All results should be confirmed by other techniques.