Understanding Yeast Two Hybrid Interactions on Leu- Medium

[higherme]

I was reading a paper on yeast two hybrid, and I was wondering why a bait protein and GAL4 activation domain show growth on Leu- medium. I thought there should be no interaction since there is no prey protein?

 

[Monique]

There can be auto activation, which is not uncommon when you only use the bait protein.

 

[higherme]

what do you mean auto activation?

 

[higherme]

is it when the bait itsef can activate transcription without the prey?

another question i want to ask is what is the differnce between doing the yeast screen on medium lacking Leu, either with glucose or with galactose? Is it because the prey protein is often induced with galactose? if there is a weak interaction, only growth shows on Leu- with galactose, whereas, in a strong interaction, both media with show growth?

thanks in advance.

 

[Monique]

is it when the bait itsef can activate transcription without the prey?

Indeed and this can lead to false positives, it is always good to check auto-activation of the bait (or use stringent conditions that rule out auto-activation).

another question i want to ask is what is the differnce between doing the yeast screen on medium lacking Leu, either with glucose or with galactose? Is it because the prey protein is often induced with galactose? if there is a weak interaction, only growth shows on Leu- with galactose, whereas, in a strong interaction, both media with show growth?

thanks in advance.

In this case you would need to use a yeast strain that is Leu-auxotroph. You do the Y2H assay on a minimal media plate (laking Leu), so that the auxotroph can not grow. Only when there is an interaction between the bait and the prey does the promoter get activated, which should drive Leu expression giving rise to growth of the yeast.