Protein protein interactions. Introduction Protein b unknown Protein a known Interaction Better...
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Transcript of Protein protein interactions. Introduction Protein b unknown Protein a known Interaction Better...
Introduction
Protein bunknown
Protein aknown
Interaction
Better understanding of protein b’s function
Introduction
Protein bFunction b
Protein aFunction a
Interaction
Detecting relationship between pathways
Detecting new pathways
Old world vs. new world
OLD WORLD
Discrete methods (1 X 1)
NEW WORLD
Comprehensive methods
MAINLY
IN THIS LECTURE
Yeast two hybrid systemSimple transcription
gene
Transcription
Complex
transcriptionDBD
DBD = DNA Binding Domain
AD
AD = Activation Domain
upstream activating sequence
UAS
Reporter gene
Transcription
Complex
transcription
UAS
DBD
bait
AD
fish
Yeast two hybrid systemHybrid proteins
Yeast two hybrid systemplasmids
Yeast
Report of the gene only in case of interaction
Between the two proteins
• Produce the yeast’s 6000 ORFs
Yeast array – producing the array
prom
oter
AD ORF
• Produce plasmids: each contains ORF + AD
• Transformation the plasmids into yeast cells
Yeast
• 2 colonies of each transformation are
inserted to the array
• ORF – Open Reading Frames
A comprehensive analysis of protein-protein interactions in S. cerevisiae P. Uetz et al..
A comprehensive analysis of protein-protein interactions in S. cerevisiae P. Uetz et al..
Yeast array – using the array
• Selection of 192 “easy” proteins
prom
oter
DBDORF
• Producing similar plasmids (DBD+protein)• Transformation the plasmids into yeast cells
YeastMATING & creatingdiploids
SELECTION OF LIVING COLONIES
BASED ON HIS3 PRODUCTION
DETECTING THE ARRAY’S PROTEIN
ACCORDING TO ITSPOSITION
Yeast array - Results over-view
• 2 undependable assays were preformed for each of the 192 proteins.
• 87 out of 192 proteins were detected as involved in protein-protein interactions (passed the 2 assays)
• total of 281 interactions were detected
Duplicates of a single DBD transformant
Activation Domain library
prom
oter
ADORF• Production of ORF+AD plasmids and transformants
prom
oter DBDORF
• Production of ORF+DBD plasmids and transformants
• Production of an AD library
The AD libraryMATING (haploids to diploids)Transferring to a selection plateDetecting the ORF’s using PCR
• 817 out of ~6000 proteins were detected as involved in protein-protein interactions
• total of 692 interactions were detected
Activation Domain library - results
Array vs. library - Comparison
ARRAYLIBRARY
TimeTakes a lot of timeRather quick
Amount of checked
interactions Plenty
(192X6000)Huge number
(6000X6000)Average number of
Interactions for an interacting protein3.31.8
Number of interactions of 12 proteins that were detected in both methods
4814
CONCLUSIONS - THE METHODS’
QUALITIES
QUICK, SIMPLER,
CHEAPER
SENSETIVE, BETTER
RESULTS
Protein arrays – producing the array
Producing the yeast’s 6000 ORF’s using plasmids
• Attaching histidine anchors to every protein
• Attaching the proteins to an array
Protein chips: from concept to practiceYoung-Sam Lee et al..
Protein chips: from concept to practiceYoung-Sam Lee et al..
Protein arrays – using the array
• Pouring a protein onto the array
Detecting the interactions
Using antibodiesthat detect the
interaction’s product
The poured Protein
is labeled
Sophisticatedassays
Mass Spectrometry of purified complexes
• Production of chimeric tagged proteins using plasmids
• The complex is isolated using the tag
• The protein creates a complex of proteins
• The complex is separated using gel
electrophoresis
• Each protein is identified by Mass Spectrometry
Mass Spectrometry of purified complexes
BENEFITS
Identifying complex interactions
Reliability can be checked
DRAWBACKS Needs specific conditions
Can lose loosely associated components
Tagging might disturb the “complexing”
Synthetic Lethal MutationsWhat’s lethal mutation?
• Examine 2 genes , “viable” and mutants
• Examining the creature carrying them
GENES
PR’ LEVELS
THE
CREATURE
Synthetic Lethal Mutations Hypothesis : these proteins are in interactions
METHOD # 1: Synthetic Lethal Mutations• Create an artificial DNA containing 2 genes with conditional mutations
• Change the conditions and detecting dead creatures
METHOD # 2: Synthetic Lethal Mutations Arrays
• Create a yeast array, every yeast contains a different mutation
MATING & creatingdiploids
STIMULATING THE CREATION OF SPORES
+SELECTIONFINDING THE DEAD
• Pour different yeasts carrying different mutations
Computational methods• Mentioned in this seminar, mainly for understanding proteins’
Functions and using to detect interactions
• Measuring mRNA levels under a variety of cellular conditions
• Grouping the genes that have similar transcriptional responses
IN VIVO, EXAMINE DIFFERENT CELL’S CONDITIONS
NOT SO ACCURATE
Computational methods Genome analysis – IN SILICO
• Genes that are consistently in the same operon, in the same order but in different and distanced creatures
ORTHOLOGS
CREATURE A
CREATURE B
Computational methods Genome analysis
• Interacting proteins have a tendency to be either present ot absent
together from fully sequenced genomes
• One gene in creature A = some genes in creature B
(ROSETTA STONE)
CREATURE A
CREATURE B
1 polypeptid
3 polypeptids