1. Protein-protein interactions Why study protein interactions? To infer function To understand regulatory networks Approach With given bait, discover target Methods Two-hybrid system Phage display

2. Two-hybrid system - 1.target plasmid Target plasmid directs transcription of the N- terminal domain of RNA polymerase α –subunit The target gene is fused in-frame to RNAPα at its the 3' end. A promoter controls transcription

3. Two-hybrid system -2. bait vector The bait vector encodes a repressor protein, to which the ‘bait’ protein of interest is fused. The promoter of the repressor protein makes expression of the bait fusion inducible by IPTG.

4. Two-hybrid system – 3. reporter system The E. coli reporter strain carries an auxotrophy (His3) and a resistance gene (Strep r ) on an F' episome. This strain is transformed with a recombinant target and a bait plasmid. Expression of target and bait protein fusions are induced by IPTG. If bait and target interact, RNApol initiates transcription of His3 and Strep r genes. Screening 1 st for His prototrophy, 2 nd for streptomycin resistance.

5. Two-hybrid system – final step Sequence the ‘positive’ target plasmid to determine the protein that interacts with a given bait

6. Phage display Cloning/reporting system is M13 phage Fusion of target proteins to either of the surface proteins g3p, g8p Propagated phages (in E.coli) express target proteins on their surface A phage library contains ~10 8 different targets

7. Phage display - screening Immobilize bait protein on magnetic beads Phages displaying target bind to the bait Bound phages are eluted and amplified in E. coli. Sequence phage DNA to determine target protein that interacted with given bait.