Chapter 18, Problem 26P

Summary Introduction

To analyze:

To detect interactions between proteins using the two-hybrid system, the following results are obtained:

A clone coding gene A gave positive results with clones B and Cwhile clone B gave positive results with clones A, D, and E but not C; and clone E gave positive results only with clone B.

Alternative clone F gave positive results with clone G but not with any of A–E. Can you explain these results?

Introduction:

The two-hybrid system is a method to determine the interaction between two proteins. The system is based on the modular nature of the transcription factor Gal4 from yeast. The two-hybrid systemactivates the downstream reporter gene. It is doneby binding of transcription factor onto the upstream activating sequence (UAS).

For Two-Hybrid system, the transcription factor divides into two parts i.e. DNA binding domains (DBD or BD) and activating domain (AD). Two fusion proteins are prepared – $\text{Gal 4 BD +}$ Bait and $\text{Gal 4 AD+}$ prey. When both fusion proteins are generated, the transcription of the gene occurs. It occurs wheninteraction of Bait part of first fusion protein and Prey part of second takes place.

Both BD and AD have different functions. The domain AD and BD is responsible for the activation of transcriptionand binding to the UASrespectively. $\text{Y2H}$-yeast two hybrid technique is used for anassessment of protein-fragment complementation.