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Yeast two-hybrid system: a genetic assay for detecting protein-protein interactions Suppose we have two proteins... X Y and the question, do these two protein bind each other? One way of answering this question is to use the yeast two hybrid method. To understand the method we must first consider how gene expression is regulated in yeast...... Yeast two-hybrid system: a genetic assay for detecting protein-protein interactions Regulation of gene expression in yeast t r a n s c r i p t i o n a c t i v a t o r a c t i v a t i o n d o m a i n t r a n s c r i p t i o n m a c h i n e r y D N A b i n d i n g d o m a i n U A S ( u p s t r e a m a c t i v a t i o n s e q u e n c e ) g e n e and now back to the question... Yeast two-hybrid system: a genetic assay for detecting protein-protein interactions Regulation of gene expression in yeast t r a n s c r i p t i o n a c t i v a t o r a c t i v a t i o n d o m a i n D N A b i n d i n g d o m a i n U A S ( u p s t r e a m a c t i v a t i o n s e q u e n c e ) t r a n s c r i p t i o n m a c h i n e r y D othese tw oprotein bind? g e n e Y X We start with yeast possessing a reporter gene (a gene making a product that is easy to detect). And now we introduce genes coding for two hybrid proteins..... r e p o r t e r g e n e Yeast two-hybrid system: a genetic assay for detecting protein-protein interactions Regulation of gene expression in yeast t r a n s c r i p t i o n a c t i v a t o r a c t i v a t i o n d o m a i n t r a n s c r i p t i o n m a c h i n e r y D N A b i n d i n g d o m a i n g e n e U A S ( u p s t r e a m a c t i v a t i o n s e q u e n c e ) Y o u r t w o h y b r i d p r o t e i n s b i n d ! yes, we have expression of the reporter indicating that the proteins bind! Y t h u s f o r m i n g a f u n c t i o n a l t r a n s c r i p t i o n a c t i v a t o r . . . X Y X X X Do these proteins bind? Y do we get expression of the reporter? r e p o r t e r g e n e e n d o f s l i d e s h o w