Lecture_12-2011 - Protein complexes - why? Proteins often...

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Protein complexes - why? Proteins often function as large, multi- subunit complexes. RNA polymerase Can get clues about the function of a protein by knowing what other proteins it contacts.
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Protein:protein interactions Genetic approach Yeast 2-hybrid Biochemical approach Co-immunoprecipitation Fusion protein affinity chromatography Cell-biology FRET - fluorescence resonance energy transfer Computational Rosetta Stone Co-regulation
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Yeast 2-hybrid approach Based on the fact some transcriptional activators have separable DNA binding domains (BD) and transcriptional activation domains (AD). GAL4, LexA
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Protein you are studying = Bait Fused to the DNA binding domain of GAL4 Protein(s) you are screening = Fish or Prey Fused to the activation domain of GAL4 Transform Bait and Fish plasmids into yeast, measure the expression of a reporter gene. Usually a gene that can be selected for when expressed.
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Image from: http://www.bioteach.ubc.ca/MolecularBiology/AYeastTwoHybridAssay/
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Yeast 2-hybrid on a genome wide scale Clone every gene in your genome into both the
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This note was uploaded on 03/01/2011 for the course MMG 433 taught by Professor Britton,r during the Spring '08 term at Michigan State University.

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Lecture_12-2011 - Protein complexes - why? Proteins often...

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