Question

Which of the following is incorrect about Yeast-two-hybrid screens?

(a) The yeast-two-hybrid system uses the transcription of a reporter gene driven by the Gal4 transcription factor to monitor whether or not two proteins are interacting

(b) The DNA-binding domain chimeric protein will not bind upstream of the reporter gene

(c) If the activation domain chimeric protein interacts with the DNA-binding domain chimeric protein, the reporter gene will be transcribed

(d) Disadvantages of the method are that only pairwise interactions are tested, and not interactions that can only take place when multiple proteins come together, as well as a high false positive rate

I have been asked this question in final exam.

My enquiry is from Protein in portion Global Approaches for Studying Protein – Protein Interactions of Bioinformatics

Answer 1

Right option is (b) The DNA-binding domain chimeric protein will not bind upstream of the reporter gene

To explain: If the interaction between two proteins, A and B, is being tested, one of their genes would be fused to the DNA-binding domain of the Gal4 transcription factor (Gal4-DBD) while the other would be fused to the activation domain (Gal4-AD). The DNA-binding domain chimeric protein will bind upstream of the reporter gene. This experiment can be carried out hundreds or even thousands of times on microassay plates, as in the case of the study by Uetz and colleagues on S.cerevisiae (yeast) interactions. Each array element on these plates contains yeast cells transformed with a particular combination of two plasmids, one carrying the DNA-binding domain chimeric protein and the other the activation domain chimeric protein.