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Protein protein interactions Introduction Protein a Protein b known unknown Interaction Better understanding of protein b’s function Introduction Protein a Protein b Function a Function b Interaction Detecting new pathways Detecting relationship between pathways Old world vs. new world OLD WORLD Discrete methods (1 X 1) MAINL Y IN THIS LECTURE NEW WORLD Comprehensive methods Yeast two hybrid system Simple transcription AD = Activation Domain DBD = DNA Binding Domain AD DBD transcription UAS upstream activating sequence gene Yeast two hybrid system Hybrid proteins bait DBD AD fish Yeast two hybrid system Hybrid proteins AD fish bait DBD transcription UAS Reporter gene Yeast two hybrid system plasmids Plasmid A DBD AD Yeast Plasmid B Yeast two hybrid system plasmids Report of the gene only in case of interaction Between the two proteins Yeast Yeast array – producing the array A comprehensive analysis of protein-protein interactions in S. cerevisiae P. Uetz et al.. • ORF – Open Reading Frames • Produce the yeast’s 6000 ORFs • Produce plasmids: each contains ORF + AD • Transformation the plasmids into yeast cells AD • 2 colonies of each transformation are inserted to the array Yeast Yeast array – producing the array ORF of protein x+ AD ORF of protein y+ AD Yeast array – using the array • Selection of 192 “easy” proteins • Producing similar plasmids (DBD+protein) • Transformation the plasmids into yeast cells SELECTION OF MATING & creating DETECTING THE LIVING COLONIES ARRAY’S diploids BASED ONPROTEIN HIS3 ACCORDING TO ITS PRODUCTION POSITION DBD Yeast Yeast array - results BEFORE (Pcf11) RNA14 AFTER RNA15 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 Activation Domain library AD • Production of ORF+AD plasmids and transformants DBD • Production of ORF+DBD plasmids and transformants • Production of an AD library Duplicates of a single DBD transformant to diploids) The MATING AD library (haploids Transferring to a selection plate Detecting the ORF’s using PCR Activation Domain library - results • 817 out of ~6000 proteins were detected as involved in protein-protein interactions • total of 692 interactions were detected Array vs. library - Comparison ARRAY LIBRARY Takes a lot of time Rather quick Plenty (192X6000) Huge number (6000X6000) Average number of Interactions for an interacting protein 3.3 1.8 Number of interactions of 12 proteins that were detected in both methods 48 14 CONCLUSIONS THE METHODS’ QUALITIES SENSETIVE, BETTER RESULTS QUICK, SIMPLER, CHEAPER Time Amount of checked interactions 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 Protein arrays – using the array • Pouring a protein onto the array Detecting the interactions The poured Protein is labeled Using antibodies that detect the interaction’s product Sophisticated assays Mass Spectrometry of purified complexes • Production of chimeric tagged proteins using plasmids • The protein creates a complex of proteins • The complex is isolated using the tag • 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 Mutations What’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 • Pour different yeasts carrying different mutations STIMULATING THE MATING & creating CREATION OF SPORES diploids FINDING THE DEAD + SELECTION 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 CREATURE A ORTHOLOGS CREATURE B Computational methods Genome analysis • Interacting proteins have a tendency to be either present ot absent together from fully sequenced genomes (ROSETTA STONE) • One gene in creature A = some genes in creature B CREATURE A CREATURE B 1 polypeptid 3 polypeptids Computational methods Genome analysis Inexpensive, fast, “widened” with the genomes DB Otology relationships are not so clear, not always reliable