Affinity chromatography/mass spec

Bait proteinGST

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Affinity chromatography/mass spec

Bait proteinGST

Add yeast extractProtein complexes bindMost proteins do not bind

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Evaluation of affinity chromatography/mass spec

Advantages:Thousands of protein complexes identifiedFunctions can be assigned to proteins

Disadvantages:False negative resultsFalse positive results

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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

Bait proteinGST

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Affinity chromatography/mass spec

False positives:• sticky proteins

Bait proteinGST

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The yeast two-hybrid system

Reporter gene

Bait proteinDNA Binding

Box 8-3Page 255

The yeast two-hybrid system

Reporter gene

Prey proteinDNA activation

Prey proteinDNA activation

Prey proteinDNA activation Prey protein

DNA activation

Box 8-3Page 255

The yeast two-hybrid system

Reporter gene

Bait proteinDNA Binding

Prey proteinDNA activation

Box 8-3Page 255

The yeast two-hybrid system

Reporter gene

Bait proteinDNA Binding

Prey proteinDNA activation

Isolate and sequence the cDNAof the binding partner you have found

Box 8-3Page 255

Evaluation of the yeast two-hybrid system

Advantages:Thousands of protein complexes identifiedFunctions can be assigned to proteins

Disadvantages:Detects only pairwise protein interactionsFalse-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

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The Rosetta Stone approach

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Marcotte et al. (1999) and other groups hypothesized that some pairs of interacting proteins are encoded by two genes in many genomes, but occasionally theyare fused into a single gene.

By scanning many genomes for examples of “fusedgenes,” several thousand protein-protein predictionshave been made.

Yeast topoisomerase II

E. coligyrase B

E. coligyrase A

The Rosetta Stone approach

Fig. 8.23Page 256

Pathway maps

A pathway is a linked set of biochemical reactions

ExPASyProNetEcoCyc: E. coli pathwaysMetaCyc: 600 pathways, 300 organismsKEGG: Kyoto Encyclopedia of Genes & Genomes

Issues:Is the extrapolation between species valid?Have orthologs been identified accurately?False positive, false negative findings

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Fig. 8.24Page 257

Fig. 8.24Page 257

Fig. 8.27Page 260

Fig. 8.29Page 262

Fig. 8.29Page 262