Chapter 18, Problem 27P

Summary Introduction

To analyze:

The two-hybrid system was used to identify interactions between proteins, and the succeeding results were obtained:

A clone coding gene A gave affirmative results with clones B and C, while clone B gave affirmative results with clones A, D, and E but not C, and clone E gave positive results only with clone B. Alternative clone F gave helpful results with clone G but not with any of $\text{A–E}$. Can you explain these results? To follow up the two-hybrid results, null loss-of-function mutations in each of genes $\text{A–G}$ were isolated. Mutants of genes A, B, C, D, and E grow at only $\text{80\%}$ of the rate of the wild type, whereas mutants of genes F and G are phenotypically impossible to differentiate from the wild type. Several double-mutant strains are constructed: The ab, ac, ad, and ae double mutants all grow at about $\text{80\%}$ of the rate of the wild type, but af and ag double mutants bear lethality.

Provide the explaination of these results. Determine the approach by which two-hybrid system and genetic interaction results complement one another. whether reconcile two-hybrid system and genetic interaction results in a single model can reconcile.

Introduction:

The two- hybrid system is a method used to determine the interaction between two proteins. The system is based on the modular nature of the transcription factor $\text{Gal4}$ from yeast. The triggering of the downstream reporter gene by binding of transcription factor onto upstream activating sequence (UAS), is the idea behind this method.

For the Two-Hybrid system, the transcription factor distributes into two domains i.e. DNA binding domain (DBD or BD) and activating domain (AD). Two fusion proteins are systematized $\text{– Gal4BD + Bait}$ and $\text{Gal4AD+}$ prey. Transcription of the gene follows when both the fusion proteins are produced and bait interacts with the prey.

Both BD and AD have different functions. BD domain boosts the binding to UAS, while AD is responsible for the activation of the transcription. $\text{Y2H}$ is a protein- fragment complementation assay.