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Protein protein interactions. Better understanding of protein b’s function. Introduction. Protein b unknown. Protein a known. Interaction. Detecting relationship between pathways. Detecting new pathways. Introduction. Protein a Function a. Protein b Function b. Interaction.

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Presentation Transcript
slide2

Better understanding of protein b’s function

Introduction

Protein b

unknown

Protein a

known

Interaction

slide3

Detecting relationship between pathways

Detecting new pathways

Introduction

Protein a

Function a

Protein b

Function b

Interaction

slide4

OLD WORLD

Discrete methods (1 X 1)

NEW WORLD

Comprehensive methods

Old world vs. new world

MAINLY

IN THIS LECTURE

slide5

AD = Activation Domain

DBD = DNA Binding Domain

AD

DBD

UAS

gene

upstream activating sequence

Yeast two hybrid system

Simple transcription

Transcription

Complex

transcription

slide6

AD

bait

DBD

fish

Yeast two hybrid system

Hybrid proteins

slide7

AD

bait

DBD

fish

UAS

Reporter gene

Yeast two hybrid system

Hybrid proteins

Transcription

Complex

transcription

slide8

Plasmid A

Plasmid B

DBD

AD

bait

fish

promoter

promoter

Yeast

Yeast two hybrid system

plasmids

slide9

Report of the gene only in case of interaction

Between the two proteins

Yeast two hybrid system

plasmids

Yeast

slide10

Yeast array – producing the array

AD

ORF

  • Produce plasmids: each contains ORF + AD

promoter

  • Transformation the plasmids into yeast cells

Yeast

A comprehensive analysis of protein-protein interactions in S. cerevisiae

P. Uetz et al..

  • ORF – Open Reading Frames
  • Produce the yeast’s 6000 ORFs
  • 2 colonies of each transformation are
  • inserted to the array
slide11

ORF of protein x+ AD

ORF of protein y+ AD

Yeast array – producing the array

slide12

Producing similar plasmids (DBD+protein)

  • Transformation the plasmids into yeast cells

DBD

ORF

promoter

Yeast

Yeast array – using the array

  • Selection of 192 “easy” proteins

MATING & creating

diploids

SELECTION OF

LIVING COLONIES

BASED ON HIS3

PRODUCTION

DETECTING THE

ARRAY’S PROTEIN

ACCORDING TO ITS

POSITION

slide13

RNA15

RNA14

Yeast array -results

BEFORE

(Pcf11)

AFTER

slide14

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
slide15

Production of ORF+AD plasmids and transformants

AD

ORF

promoter

  • Production of ORF+DBD plasmids and transformants

Duplicates of a single DBD transformant

DBD

ORF

promoter

The AD library

Activation Domain library

  • Production of an AD library

MATING (haploids to diploids)

Transferring to a selection plate

Detecting the ORF’s using PCR

slide16

Activation Domain library - results

  • 817 out of ~6000 proteins were detected as involved in protein-protein interactions
  • total of 692 interactions were detected
slide17

Array vs. library - Comparison

SENSETIVE, BETTER RESULTS

QUICK, SIMPLER, CHEAPER

slide18

Protein arrays – producing the array

Protein chips: from concept to practice

Young-Sam Lee et al..

  • Producing the yeast’s 6000 ORF’s using plasmids
  • Attaching histidine anchors to every protein
  • Attaching the proteins to an array
slide19

The poured

Protein

is labeled

Using antibodies

that detect the

interaction’s product

Sophisticated

assays

Detecting the interactions

Protein arrays – using the array

  • Pouring a protein onto the array
slide20

Production of chimeric tagged proteins using plasmids

  • The protein creates a complex of proteins
  • The complex is separated using gel
  • electrophoresis

Mass Spectrometry of purified complexes

  • The complex is isolated using the tag
  • Each protein is identified by Mass Spectrometry
slide21

BENEFITS

Identifying complex interactions

Reliability can be checked

DRAWBACKS

Needs specific conditions

Can lose loosely associated components

Tagging might disturb the “complexing”

Mass Spectrometry of purified complexes

slide22

Examine 2 genes , “viable”

and mutants

GENES

PR’ LEVELS

LIVES

LIVES

THE

CREATURE

LIVES

DEAD

Synthetic Lethal Mutations

What’s lethal mutation?

  • Examining the creature carrying them
slide23

Create a yeast array, every yeast contains a different mutation

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

  • Pour different yeasts carrying different mutations

MATING & creating

diploids

STIMULATING THE

CREATION OF SPORES

+ SELECTION

FINDING THE DEAD

slide24

IN VIVO, EXAMINE DIFFERENT CELL’S CONDITIONS

NOT SO ACCURATE

Computational methods

  • Mentioned in this seminar, mainly for understanding proteins’
  • Functions and using to detect interactions

Correlated mRNA expression

  • Measuring mRNA levels under a variety of cellular conditions
  • Grouping the genes that have similar transcriptional responses
slide25

CREATURE A

ORTHOLOGS

CREATURE B

Computational methods

Genome analysis – IN SILICO

Prokaryotic\'s operons

  • Genes that are consistently in the same operon, in the same order but in different and distanced creatures
slide26

CREATURE A

1 polypeptid

HOMOLOGY

CREATURE B

3 polypeptids

Computational methods

Genome analysis

Phylogenetic profile

  • Interacting proteins have a tendency to be either present ot absent
  • together from fully sequenced genomes

Gene fusion

(ROSETTA STONE)

  • One gene in creature A = some genes in creature B
slide27

Inexpensive, fast, “widened” with the genomes DB

Computational methods

Genome analysis

Otology relationships are not so clear,

not always reliable

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