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Affinity chromatography/mass spec GST Bait protein Page 252 Affinity chromatography/mass spec GST Bait protein Add yeast extract Protein complexes bind Most proteins do not bind Page 252 Evaluation of affinity chromatography/mass spec Advantages: Thousands of protein complexes identified Functions can be assigned to proteins Disadvantages: False negative results False positive results Page 253-254 Affinity chromatography/mass spec False negatives: • Bait must be properly localized and in its native condition • Affinity tag may interfere with function • Transient protein interactions may be missed • Highly specific physiological conditions may be required • Bias against hydrophobic, and small proteins GST Bait protein Page 253 Affinity chromatography/mass spec False positives: • sticky proteins GST Bait protein Page 253 The yeast two-hybrid system Bait protein DNA Binding Reporter gene Box 8-3 Page 255 The yeast two-hybrid system Prey protein DNA activation Prey protein DNA activation Reporter gene Prey protein DNA activation Prey protein DNA activation Box 8-3 Page 255 The yeast two-hybrid system Bait protein DNA Binding Prey protein DNA activation Reporter gene Box 8-3 Page 255 The yeast two-hybrid system Bait protein DNA Binding Prey protein DNA activation Reporter gene Isolate and sequence the cDNA of the binding partner you have found Box 8-3 Page 255 Evaluation of the yeast two-hybrid system Advantages: Thousands of protein complexes identified Functions can be assigned to proteins Disadvantages: Detects only pairwise protein interactions False-negative results (as for affinity chromatography) -- bait may be mislocalized -- transient interactions may be missed -- some complexes require special conditions -- bias against hydrophobic proteins False-positive results -- some proteins may be sticky -- bait protein may auto-activate a reporter Page 256 The Rosetta Stone approach Marcotte et al. (1999) and other groups hypothesized that some pairs of interacting proteins are encoded by two genes in many genomes, but occasionally they are fused into a single gene. By scanning many genomes for examples of “fused genes,” several thousand protein-protein predictions have been made. Page 258 The Rosetta Stone approach Yeast topoisomerase II E. coli gyrase B E. coli gyrase A Fig. 8.23 Page 256 Pathway maps A pathway is a linked set of biochemical reactions ExPASy ProNet EcoCyc: E. coli pathways MetaCyc: 600 pathways, 300 organisms KEGG: Kyoto Encyclopedia of Genes & Genomes Issues: Is the extrapolation between species valid? Have orthologs been identified accurately? False positive, false negative findings Page 258 Fig. 8.24 Page 257 Fig. 8.24 Page 257 Fig. 8.27 Page 260 Fig. 8.29 Page 262 Fig. 8.29 Page 262
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