The "Two-hybrid System" for detecting protein-protein interactions in yeast An easy and powerful system for detecting noncovalent protein-protein interactions in vivo in yeast was developed independently by Stan Fields and Roger Brent about 10 years ago. Since that time many researchers have discovered and reported new connections between proteins using their techniques. Since many biological processes are carried out in protein complexes formed by noncovalent bonds, knowledge about proteinprotein interactions is extremely useful to understand how these complexes are structured and how they work. The principle of the experiment is based on the modular nature of transcription factors. Certain transcription activating proteins act by (1) binding to a special DNA sequence near the promoter, and (2) doing something that is still mysterious to activate transcription from the promoter. The part of the transcriptional activating protein that binds DNA (the DNA binding domain or DBD) can be separated from the rest of the protein and will still bind to DNA, but will not activate transcription. The activating part of the protein can also be separated and attached to a different DBD, and will activate transcription of a different set of promoters, but will not activate transcription if it is not attached to a DBD. The Fields system relies on the GAL4 transcriptional activator (which contains both a DBD and an activation domain), whereas the Brent system relies on nonyeast proteins like E. coli lexA (for its DBD) and Herpesvirus VP16 (for its activation domain). The expression of a DBD covalently fused to an activation domain (as it is in the normal GAL4 protein) leads to expression of genes to which the DBD can bind (In wild type yeast, the GAL genes). The normal GAL genes are not convenient to assay in terms of their expression so we use “reporter genes” whose activity is easy to see. The expression of these "reporter genes" report that the transcription factor is functioning. If a separate DBD and a separate activation domain are expressed in the same cell and they do not associate, no functional activator protein is made and no reporter gene expression is detected. This allows for testing for noncovalent interactions as follows. If the DBD is joined to a protein of interest X and the activation domain is joined to a protein Y thought to bind noncovalently to X, and these two, hybrid proteins (get it?, two-hybrid) are both expressed in the same cell, they can bind and form a protein complex that both binds DNA and activates transcription (even though X and Y may have nothing to do with transcription, here they only serve to bring together the DBD and the activation domain). Looking for signs of reporter expression will tell whether X and Y associate. Notice that an absence of reporter function does not mean that X and Y do not associate. A negative result cannot be interpreted (As Carl Sagan was fond of saying about aliens, "Absence of evidence is not evidence of absence."). The fusion protein could be unstable, or not correctly folded or have no DNA binding activity or activation activity for other reasons, or might not be transported to the nucleus, etc., even though in real life, X and Y might be associated tightly. Only a positive signal can be taken as a sign of association. For the same reasons it is difficult to use the amount of reporter expression as an indication of the strength of the interaction. The proteins of interest X and Y can come from any source; they need not be yeast proteins. In fact many studies using the two-hybrid system test mammalian cell proteins. It is also possible to search for new proteins that interact with a protein of interest X by screening libraries of sequences fused to the activation domain. Reporter expression reveals members of the library that interact with X and these can be recovered and characterized. Today we will begin a demonstration experiment using the two-hybrid system of Fields to test for interactions between and among numerous different yeast splicing proteins that are involved in recognition of the pre-mRNA branchpoint region and the addition of the U2 snRNP to the assembling spliceosome. Outline of the Experiment The class will be provided with plasmids encoding two-hybrid proteins made by fusing yeast splicing factor genes to the DBD and/or the activation domain of GAL4. There are 18 plasmids, 9 of them (the pAS plasmids) will be transformed into yeast strain Y187 using the TRP selectable marker on pAS, and the other 9 (the pACT plasmids) will go into Y190 using the LEU selectable marker on the pACT plasmids. Transformants of each Y187 strain will be mated to transformants from every Y190 strain on a 9 by 9 grid, selecting for diploids that carry both plasmids on YMD-leu, -trp. After mating the diploids will be replica plated to YMD-his +25mM 3-AT (3-amino-1, 2, 4, triazole is an inhibitor of the HIS3 enzyme and is necessary because the reporter is "leaky") for testing expression of a HIS reporter, and to YMD-leu,-trp for a beta-galactosidase assay to test expression from a lacz reporter. 3-amino-1,2,4-triazole (3-AT) competitively inhibits imidazole glycerol phosphate dehydratase, a His biosynthetic enzyme (Hilton et al., 1965; Klopotowski and Wiater, 1965; Wiater et al., 1971), and therefore can limit histidine biosynthesis and growth. For twohybrid screens that use a HIS3 reporter, 3-AT is employed – the HIS3 gene encodes the enzyme activity inhibited by 3AT. High expression of this reporter gene, which arises from a successful two-hybrid interaction, can overcome the growth-inhibitory effect of 3-AT in the medium. When 3-AT is added to yeast media (1-10 mM) it will limit histidine biosynthesis and is used in two-hybrid screens to "fine tune" leaky expression of the HIS3 reporter gene.Therefore, the use of 3-AT and the HIS3 reporter enables positive selection for successful two-hybrid interactions. Reporter expression will show which splicing proteins interact with which others. The proteins we are testing are BBP, CUS1, CUS2, HSH49, HSH155, MUD2, PRP9, PRP11, PRP21, SUB2, and as a control, URA3. On Day 1 we will transform Y187 with the pAS plasmids to Trp independences, and Y190 with the pACT plasmids to leucine independence. Everyone should do at least one and the plates should be incubated at 30°C. I may ask some of you to do more than one. You will use transformation method 2, except that the competent cells will have already been prepared The following lab day, assuming all the transformations went as planned, each team will streak two YPAD plates, one plate with 5 of the Y187 transformants (one of each of 5 plasmids) and one plate with each of 5 of the Y190 transformants. After these grow overnight, they are stamped on the same velveteen at right angles, and then to a YPAD plate for mating as we did before. These two strains did not mate that well last year, so we should let them grow together at least overnight. Then the YPAD plate should be replicated to an SCD-trp-leu plate to select for Y187/Y190 diploids. We will need to make sure we have all strains containing all combinations of the pairs of plasmids. This SCD-leu-trp mating selection plate will be the master plate to make replicas for the beta gal assay, and to an SCDhis+3AT plate to test for HIS+ growth due to activation of the HIS reporter. We will check growth on SCD-his+3AT, and after the replicated SCD-trpleu plate grows, we will overlay it with agarose containing X-gal, to assay for betagalactosidase enzyme, the product of the lacZ reporter. Genotypes of Y187 and Y190 Y187 MATα, ura3-52, his3-200, ade2-101, trp1-901, leu2-3, 112, gal4Δ, met-, gal80Δ, URA3::GAL1UAS-GAL1TATA-lacz Y190 MATa, ura3-52, his3-200, lys2-801, ade2-101, trp1-901, leu2-3, 112, gal4Δ, met-, gal80Δ, LYS2::GAL1UAS-HIS3TATA-HIS3, URA3::GAL1UAS-GAL1TATA-lacz YM Plates/media YM Media Ingredients Amount 1 Yeast Nitrogen Base 6.7 g (without amino acids) Bacto-Agar 20 g (Wash before using) Water 900 ml 2 Separately sterilize Glucose 20 g Water 100 ml Autoclave solutions 1 and 2 separately Mix the two thoroughly and then pour plates (25 ml per plate) 5xTEL 0.5M LiAc 50 mM Tris HCl pH 7.5 5 mM EDTA Final volume Filter sterilize and store RT 50ml of 1M stock 5ml of 1M stock 1ml of 0.5M stock 100ml 1xTEL Dilute the 5x TEL with sterile water to 1x and use 50% PEG 4000 Dissolve 50g PEG4000 in 100 ml water. Filter sterilize and store PEG/TEL 80ml 50% PEG4000 20 ml 5xTEL Salmon sperm DNA Dissolve in water to 10 mg/ml Sonicate 1 min. x3 boil 10 min. and aliquot. Protocol Grow strain in 10 ml YPD overnight Next day measure OD 600 Inoculate 100ml YPD with overnight culture so that OD is 0.1/ml (i.e. 10 OD of cells) Grow till OD = 0.8 30C Spin down cell 3 min 3000 rpm Resuspend 10 ml 1xTEL and shake overnight at Room temp Next day spin down cells 3 min 3000 rpm Resuspend in 1 ml 1xTEL. Leave 30 min. on the bench at Room temp. In a eppendorf tube add Sonicated Salmon Sperm DNA (10 mg/ml) 5 ul DNA (0.2 ug) 5 ul Competent Cells 100 ul Mix by pipeting up and down Incubate room temp without shaking for 30 min. Add 700 ul 40% PEG/TEL Mix by pipeting up and down Incubate room temp without shaking for 60 min. Heat shock 42C 10 min. Spin cells gently (setting 7 in Eppendorf microfuge) for 10 sec. Resuspend cells in 1 ml sterile water and plate on plates selecting for the plasmid. You want to get about 200 colonies per plate. Grow 30C for two days. LacZ overlay assay Grow your yeast on selective plates until colonies are 1.5-2 mm or so. Melt a solution of 1% agarose by microwaving at low power (TAKE OFF THE CAP!). Cool to 55°C in the water bath. Prewarm some 1 M Na PO4 pH7.0 to 55°C. Prewarm a 50 ml plastic test tube. You will need 5 ml of 1% agarose, 5 ml of Na PO4 pH7.0, 100 ul 10% SDS, and 200 ul of 10% X-gal in dimethyl formamide (DMF) per 90 mm petri dish. (NB less X-gal will work for strong expressing clones, but KEEP the DMF amount constant!) SDS and DMF are essential for permiablizing the yeast cells. Mix components in the following way to avoid precipitation: Put 5 ml molten agarose into the prewarmed test tube. Add 100 ul SDS, swirl to mix. Put 5 ml prewarmed Na PO4 pH7.0 into the agarose-SDS and mix. Add 200 ul X-gal in DMF, mix. With a disposable pipette, draw as much of the molten mixture up as possible and carefully apply it to the surface of the petri plate. Adding it too fast may disturb the yeast on the plate, too slowly and the mixture may solidify. After the overlaid mixture solidifies (a couple of minutes), put the plate at 37°C. Strong lacZ expressing strains will turn blue within an hour. Plates can be incubated longer to detect weaker lacZ expression. It is possible to use less X-gal, but keep the DMF constant (e. g. can use 200 ul of a 5% solution of X-gal in DMF).